Amino lipids and methods for the delivery of nucleic acids

ABSTRACT

The present invention provides superior compositions and methods for the delivery of therapeutic agents to cells. In particular, these include novel lipids and nucleic acid-lipid particles that provide efficient encapsulation of nucleic acids and efficient delivery of the encapsulated nucleic acid to cells in vivo. The compositions of the present invention are highly potent, thereby allowing effective knock-down of specific target proteins at relatively low doses. In addition, the compositions and methods of the present invention are less toxic and provide a greater therapeutic index compared to compositions and methods previously known in the art.

CROSS-REFERENCE(S) TO RELATED APPLICATION(S)

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Patent Application No. 61/104,219 filed Oct. 9, 2008; U.S.Provisional Patent Application No. 61/104,212 filed Oct. 9, 2008; andU.S. Provisional Patent Application No. 61/220,666 filed Jun. 26, 2009,where these (three) provisional applications are incorporated herein byreference in their entireties.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 480208_(—)461PC—SEQUENCE_LISTING.txt. The textfile is 9 KB, was created on Oct. 9, 2009, and is being submittedelectronically via EFS-Web.

BACKGROUND

1. Technical Field

The present invention relates to the field of therapeutic agent deliveryusing lipid particles. In particular, the present invention providescationic lipids and lipid particles comprising these lipids, which areadvantageous for the in vivo delivery of nucleic acids, as well asnucleic acid-lipid particle compositions suitable for in vivotherapeutic use. Additionally, the present invention provides methods ofmaking these compositions, as well as methods of introducing nucleicacids into cells using these compositions, e.g., for the treatment ofvarious disease conditions.

2. Description of the Related Art

Therapeutic nucleic acids include, e.g., small interfering RNA (siRNA),micro RNA (miRNA), antisense oligonucleotides, ribozymes, plasmids, andimmune stimulating nucleic acids. These nucleic acids act via a varietyof mechanisms. In the case of siRNA or miRNA, these nucleic acids candown-regulate intracellular levels of specific proteins through aprocess termed RNA interference (RNAi). Following introduction of siRNAor miRNA into the cell cytoplasm, these double-stranded RNA constructscan bind to a protein termed RISC. The sense strand of the siRNA ormiRNA is displaced from the RISC complex providing a template withinRISC that can recognize and bind mRNA with a complementary sequence tothat of the bound siRNA or miRNA. Having bound the complementary mRNAthe RISC complex cleaves the mRNA and releases the cleaved strands. RNAican provide down-regulation of specific proteins by targeting specificdestruction of the corresponding mRNA that encodes for proteinsynthesis.

The therapeutic applications of RNAi are extremely broad, since siRNAand miRNA constructs can be synthesized with any nucleotide sequencedirected against a target protein. To date, siRNA constructs have shownthe ability to specifically down-regulate target proteins in both invitro and in vivo models. In addition, siRNA constructs are currentlybeing evaluated in clinical studies.

However, two problems currently faced by siRNA or miRNA constructs are,first, their susceptibility to nuclease digestion in plasma and, second,their limited ability to gain access to the intracellular compartmentwhere they can bind RISC when administered systemically as the freesiRNA or miRNA. These double-stranded constructs can be stabilized byincorporation of chemically modified nucleotide linkers within themolecule, for example, phosphothioate groups. However, these chemicalmodifications provide only limited protection from nuclease digestionand may decrease the activity of the construct. Intracellular deliveryof siRNA or miRNA can be facilitated by use of carrier systems such aspolymers, cationic liposomes or by chemical modification of theconstruct, for example by the covalent attachment of cholesterolmolecules [reference]. However, improved delivery systems are requiredto increase the potency of siRNA and miRNA molecules and reduce oreliminate the requirement for chemical modification.

Antisense oligonucleotides and ribozymes can also inhibit mRNAtranslation into protein. In the case of antisense constructs, thesesingle stranded deoxynucleic acids have a complementary sequence to thatof the target protein mRNA and can bind to the mRNA by Watson-Crick basepairing. This binding either prevents translation of the target mRNAand/or triggers RNase H degradation of the mRNA transcripts.Consequently, antisense oligonucleotides have tremendous potential forspecificity of action (i.e., down-regulation of a specificdisease-related protein). To date, these compounds have shown promise inseveral in vitro and in vivo models, including models of inflammatorydisease, cancer, and HIV (reviewed in Agrawal, Trends in Biotech.14:376-387 (1996)). Antisense can also affect cellular activity byhybridizing specifically with chromosomal DNA. Advanced human clinicalassessments of several antisense drugs are currently underway. Targetsfor these drugs include the bcI2 and apolipoprotein B genes and mRNAproducts.

Immune-stimulating nucleic acids include deoxyribonucleic acids andribonucleic acids. In the case of deoxyribonucleic acids, certainsequences or motifs have been shown to illicit immune stimulation inmammals. These sequences or motifs include the CpG motif,pyrimidine-rich sequences and palindromic sequences. It is believed thatthe CpG motif in deoxyribonucleic acids is specifically recognized by anendosomal receptor, toll-like receptor 9 (TLR-9), which then triggersboth the innate and acquired immune stimulation pathway. Certain immunestimulating ribonucleic acid sequences have also been reported. It isbelieved that these RNA sequences trigger immune activation by bindingto toll-like receptors 6 and 7 (TLR-6 and TLR-7). In addition,double-stranded RNA is also reported to be immune stimulating and isbelieve to activate via binding to TLR-3.

One well known problem with the use of therapeutic nucleic acids relatesto the stability of the phosphodiester internucleotide linkage and thesusceptibility of this linker to nucleases. The presence of exonucleasesand endonucleases in serum results in the rapid digestion of nucleicacids possessing phosphodiester linkers and, hence, therapeutic nucleicacids can have very short half-lives in the presence of serum or withincells. (Zelphati, O., et al., Antisense. Res. Dev. 3:323-338 (1993); andThierry, A. R., et al., pp 147-161 in Gene Regulation: Biology ofAntisense RNA and DNA (Eds. Erickson, R P and Izant, J G; Raven Press,NY (1992)). Therapeutic nucleic acid being currently being developed donot employ the basic phosphodiester chemistry found in natural nucleicacids, because of these and other known problems.

This problem has been partially overcome by chemical modifications thatreduce serum or intracellular degradation. Modifications have beentested at the internucleotide phosphodiester bridge (e.g., usingphosphorothioate, methylphosphonate or phosphoramidate linkages), at thenucleotide base (e.g., 5-propynyl-pyrimidines), or at the sugar (e.g.,2′-modified sugars) (Uhlmann E., et al. Antisense: ChemicalModifications. Encyclopedia of Cancer, Vol. X., pp 64-81 Academic PressInc. (1997)). Others have attempted to improve stability using 2′-5′sugar linkages (see, e.g., U.S. Pat. No. 5,532,130). Other changes havebeen attempted. However, none of these solutions have proven entirelysatisfactory, and in vivo free therapeutic nucleic acids still have onlylimited efficacy.

In addition, as noted above relating to siRNA and miRNA, problems remainwith the limited ability of therapeutic nucleic acids to cross cellularmembranes (see, Vlassov, et al., Biochim. Biophys. Acta 1197:95-1082(1994)) and in the problems associated with systemic toxicity, such ascomplement-mediated anaphylaxis, altered coagulatory properties, andcytopenia (Galbraith, et al., Antisense Nucl. Acid Drug Des. 4:201-206(1994)).

To attempt to improve efficacy, investigators have also employedlipid-based carrier systems to deliver chemically modified or unmodifiedtherapeutic nucleic acids. In Zelphati, O. and Szoka, F. C., J. Contr.Rel. 41:99-119 (1996), the authors refer to the use of anionic(conventional) liposomes, pH sensitive liposomes, immunoliposomes,fusogenic liposomes, and cationic lipid/antisense aggregates. SimilarlysiRNA has been administered systemically in cationic liposomes, andthese nucleic acid-lipid particles have been reported to provideimproved down-regulation of target proteins in mammals includingnon-human primates (Zimmermann et al., Nature 441: 111-114 (2006)).

In spite of this progress, there remains a need in the art for improvednucleic acid-lipid particles and compositions that are suitable forgeneral therapeutic use. Preferably, these compositions wouldencapsulate nucleic acids with high-efficiency, have high drug:lipidratios, protect the encapsulated nucleic acid from degradation andclearance in serum, be suitable for systemic delivery, and provideintracellular delivery of the encapsulated nucleic acid. In addition,these nucleic acid-lipid particles should be well-tolerated and providean adequate therapeutic index, such that patient treatment at aneffective dose of the nucleic acid is not associated with significanttoxicity and/or risk to the patient. The present invention provides suchcompositions, methods of making the compositions, and methods of usingthe compositions to introduce nucleic acids into cells, including forthe treatment of diseases.

BRIEF SUMMARY

The present invention provides novel amino lipids, as well as lipidparticles comprising the same. These lipid particles may furthercomprise an active agent and be used according to related methods of theinvention to deliver the active agent to a cell.

In one embodiment, the present invention includes an amino lipid havingthe following structure (I):

or salts thereof, wherein

R¹ and R² are either the same or different and independently optionallysubstituted C₁₂-C₂₄ alkyl, optionally substituted C₁₂-C₂₄ alkenyl,optionally substituted C₁₂-C₂₄ alkynyl, or optionally substitutedC₁₂-C₂₄ acyl;

R³ and R⁴ are either the same or different and independently optionallysubstituted C₁-C₆alkyl, optionally substituted C₁-C₆alkenyl, oroptionally substituted C₁-C₆alkynyl or R³ and R⁴ may join to form anoptionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or2 heteroatoms chosen from nitrogen and oxygen;

R⁵ is either absent or hydrogen or C₁-C₆ alkyl to provide a quaternaryamine;

m, n, and p are either the same or different and independently either 0or 1 with the proviso that m, n, and p are not simultaneously 0;

q is 2, 3, or 4; and

Y and Z are either the same or different and independently O, S, or NH.

In one embodiment, the amino lipid is the amino lipid having structure(I) wherein q is 2.

In certain embodiments, the amino lipid has the following structure(II):

or salts thereof, wherein

R¹ and R² are either the same or different and independently optionallysubstituted C₁₂-C₂₄ alkyl, optionally substituted C₁₂-C₂₄ alkenyl,optionally substituted C₁₂-C₂₄ alkynyl, or optionally substitutedC₁₂-C₂₄ acyl;

R³ and R⁴ are either the same or different and independently optionallysubstituted C₁-C₆ alkyl, optionally substituted C₁-C₆ alkenyl, oroptionally substituted C₁-C₆ alkynyl or R³ and R⁴ may join to form anoptionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or2 heteroatoms chosen from nitrogen and oxygen;

R⁵ is either absent or is hydrogen or C₁-C₆ alkyl to provide aquaternary amine;

m, n, and p are either the same or different and independently either 0or 1 with the proviso that m, n, and p are not simultaneously 0;

Y and Z are either the same or different and independently O, S, or NH.

In particular embodiments, the amino lipid has the following structure(III):

wherein

n is 2, 3, or 4.

In one particular embodiment, the amino lipid has the structure:

In one particular embodiment, the amino lipid has the structure:

In one particular embodiment, the amino lipid has the structure:

In another embodiment, the present invention provides an amino lipidhaving the structure:

In further related embodiments, the present invention includes a lipidparticle comprising one or more of the above amino lipids of the presentinvention. In certain embodiments, the particle further comprises aneutral lipid and a lipid capable of reducing particle aggregation. Inone particular embodiment, the lipid particle consists essentially of:(i) DLin-K-C2-DMA; (ii) a neutral lipid selected from DSPC, POPC, DOPE,and SM; (iii) cholesterol; and (iv) PEG-S-DMG, PEG-C-DOMG or PEG-DMA, ina molar ratio of about 20-60% DLin-K-C2-DMA:5-25% neutral lipid:25-55%Chol:0.5-15% PEG-S-DMG, PEG-C-DOMG or PEG-DMA. In one particularembodiment, the lipid particle consists essentially of: (i) DLin-K²-DMA;(ii) a neutral lipid selected from DSPC, POPC, DOPE, and SM; (iii)cholesterol; and (iv) PEG-S-DMG, PEG-C-DOMG or PEG-DMA, in a molar ratioof about 20-60% DLin-K²-DMA:5-25% neutral lipid:25-55% Chol:0.5-15%PEG-S-DMG, PEG-C-DOMG or PEG-DMA.

In additional related embodiments, the present invention includes lipidparticles of the invention that further comprise a therapeutic agent. Inone embodiment, the therapeutic agent is a nucleic acid. In variousembodiments, the nucleic acid is a plasmid, an immunostimulatoryoligonucleotide, a siRNA, a microRNA, an antisense oligonucleotide, or aribozyme.

In yet another related embodiment, the present invention includes apharmaceutical composition comprising a lipid particle of the presentinvention and a pharmaceutically acceptable excipient, carrier, ordiluent.

The present invention further includes, in other related embodiments, amethod of modulating the expression of a polypeptide by a cell,comprising providing to a cell a lipid particle or pharmaceuticalcomposition of the present invention. In particular embodiments, thelipid particle comprises a therapeutic agent selected from an siRNA, amicroRNA, an antisense oligonucleotide, and a plasmid capable ofexpressing an siRNA, a microRNA, or an antisense oligonucleotide, andwherein the siRNA, microRNA, or antisense RNA comprises a polynucleotidethat specifically binds to a polynucleotide that encodes thepolypeptide, or a complement thereof, such that the expression of thepolypeptide is reduced. In another embodiment, the nucleic acid is aplasmid that encodes the polypeptide or a functional variant or fragmentthereof, such that expression of the polypeptide or the functionalvariant or fragment thereof is increased.

In yet a further related embodiment, the present invention includes amethod of treating a disease or disorder characterized by overexpressionof a polypeptide in a subject, comprising providing to the subject alipid particle or pharmaceutical composition of the present invention,wherein the therapeutic agent is selected from an siRNA, a microRNA, anantisense oligonucleotide, and a plasmid capable of expressing an siRNA,a microRNA, or an antisense oligonucleotide, and wherein the siRNA,microRNA, or antisense RNA comprises a polynucleotide that specificallybinds to a polynucleotide that encodes the polypeptide, or a complementthereof.

In another related embodiment, the present invention includes a methodof treating a disease or disorder characterized by underexpression of apolypeptide in a subject, comprising providing to the subject thepharmaceutical composition of the present invention, wherein thetherapeutic agent is a plasmid that encodes the polypeptide or afunctional variant or fragment thereof.

In a further embodiment, the present invention includes a method ofinducing an immune response in a subject, comprising providing to thesubject the pharmaceutical composition of the present invention, whereinthe therapeutic agent is an immunostimulatory oligonucleotide. Inparticular embodiments, the pharmaceutical composition is provided tothe patient in combination with a vaccine or antigen.

In a related embodiment, the present invention includes a vaccinecomprising the lipid particle of the present invention and an antigenassociated with a disease or pathogen. In one embodiment, the lipidparticle comprises an immunostimulatory nucleic acid or oligonucleotide.In a particular embodiment, the antigen is a tumor antigen. In anotherembodiment, the antigen is a viral antigen, a bacterial antigen, or aparasitic antigen.

The present invention further includes methods of preparing the lipidparticles and pharmaceutical compositions of the present invention, aswell as kits useful in the preparation of these lipid particle andpharmaceutical compositions.

In particle embodiments, any of the compositions or methods of thepresent invention may comprise any of the other cationic lipids of thepresent invention, as described herein, as the cationic lipid. Inparticular embodiments, the cationic lipid is DLin-K²-DMA orDLin-K6-DMA.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a diagram of a proposed mechanism of action for the membranedisruptive effects of cationic lipids and a structural diagram ofDLinDMA divided into headgroup, linker and hydrocarbon chain domains. Inisolation, cationic lipids and endosomal membrane anionic lipids such asphosphatidylserine adopt a cylindrical molecular shape, which iscompatible with packing in a bilayer configuration. However, whencationic and anionic lipids are mixed together, they combine to form anion pairs where the cross-sectional area of the combined headgroup isless than that of the sum of individual headgroup areas in isolation.The ion pair therefore adopts a molecular “cone” shape, which promotesthe formation of inverted, non-bilayer phases such as the hexagonalH_(II) phase illustrated. Inverted phases do not support bilayerstructure and are associated with membrane fusion and membranedisruption (Hafez, I. M., et al., Gene Ther 8, 1188-1196 (2001) andCullis, P. R., et al., Chem Phys Lipids 40, 127-144 (1986)).

FIGS. 2A-B are graphs depicting the in vivo silencing activity ofnucleic acid-lipid particles comprising various cationic lipids. FIG. 2Adepicts the silencing activity of DLinDAP (▾), DLinDMA (▴), DLin-K-DMA(▪) and DLin-K-C2-DMA (●) formulations in the mouse FVII model. Allnucleic acid-lipid particles were prepared using the preformed vesicle(PFV) method and were composed of ionizable cationic lipid, DSPC,cholesterol and PEG-lipid (40/10/40/10 mol/mol) with a FVIIsiRNA-to-total lipid ratio of ˜0.05 (wt/wt). Data points are expressedas a percentage of PBS control animals and represent group mean(n=5)±s.d, and all formulations were compared within the same study.FIG. 2B demonstrates the influence of headgroup extensions on theactivity of DLin-K-DMA. DLin-K-DMA (▪) had additional methylene groupsadded between the DMA headgroup and the ketal ring linker to generateDLin-K-C2-DMA (●), DLin-K-C3-DMA (▴) and DLin-K-C4-DMA (▾). The activityof PFV formulations of each lipid was assessed in the mouse FVII model.Data points are expressed as a percentage of PBS control animals andrepresent group mean (n=4)±s.d.

FIG. 3 is a graph depicting the amount of residual FVII followingadministration of various dosages of the indicated nucleic acid-lipidparticle formulations comprising encapsulated FVII siRNA to mice.

FIG. 4 is a graph depicting the amount of residual FVII followingadministration of various dosages of the indicated nucleic acid-lipidparticle formulations comprising encapsulated FVII siRNA to rats.

FIG. 5 is a graph comparing the amount of residual FVII followingadministration of two nucleic acid-lipid particle formulations(DLin-K-C2-DMA or DLin-K-DMA) comprising encapsulated FVII siRNA to miceor rats.

FIG. 6 is a graph comparing the amount of residual FVII followingadministration of various concentrations of three different nucleicacid-lipid particle formulations (DLin-K6-DMA, DLin-K-C2-DMA, andDLin-K-DMA) comprising encapsulated FVII siRNA to mice.

FIG. 7 is a graph depicting the amount of residual FVII followingadministration of various dosages of the indicated nucleic acid-lipidparticle formulations comprising encapsulated FVII si RNA to animals. C2indicates DLin-K-C2-DMA; C3 indicates DLin-K-C3-DMA; and C4 indicatesDLin-K-C4-DMA.

FIG. 8 is a graph showing the amount of residual FVII followingadministration of various dosages of the nucleic acid-lipid particleformulations comprising the different indicated cationic lipids: DLinDAP(•), DLinDMA (▴), DLin-K-DMA (▪), or DLIN-K-C2-DMA (♦).

FIGS. 9A-B illustrate the efficacy of KC2-SNALP formulations. FIG. 9A isa graph showing the improved efficacy of KC2-SNALP versus aDLin-K-C2-DMA PFV formulation in mice. The in vivo efficacy of KC2-SNALP(◯) was compared to that of the un-optimized DLin-KC2-DMA PFVformulation (●) in the mouse FVII model. Data points are expressed as apercentage of PBS control animals and represent group mean (n=5)±s.d.FIG. 9B depicts the efficacy of KC2-SNALP in non-human primates.Cynomolgus monkeys (n=3 per group) received either 0.03, 0.1, 0.3 or 1mg/kg siTTR, or 1 mg/kg siApoB formulated in KC2-SNALP or PBS as 15minute intravenous infusions (5 mL/kg) via the cephalic vein. Animalswere sacrificed at 48 hours post-administration. TTR mRNA levelsrelative to GAPDH mRNA levels were determined in liver samples. Datapoints represent group mean±s.d. *=P<0.05; **=P<0.005.

DETAILED DESCRIPTION

The present invention is based, in part, upon the identification ofnovel cationic lipids that provide superior results when used in lipidparticles for the in vivo delivery of a therapeutic agent. Inparticular, the present invention provides nucleic acid-lipid particlecompositions (also referred to as formulations or liposomalformulations) comprising a cationic lipid according to the presentinvention that provide increased activity of the nucleic acid andsignificant tolerability of the compositions in vivo, which is expectedto correlate with a significant increase in therapeutic index ascompared to nucleic acid-lipid particle compositions previouslydescribed.

As described in the accompanying Examples, a rational design approachwas employed for the discovery of novel lipids for use innext-generation lipid particle systems to deliver nucleic acids,including, e.g., RNAi therapeutics. Using this approach, importantstructure-activity considerations for ionizable cationic lipids weredescribed, and multiple lipids based on the DLinDMA structure weredesigned and characterized. Nucleic acid-lipid particles comprising thecationic lipid termed DLin-K-C2-DMA were shown to be well-tolerated inboth rodent and non-human primates and exhibited in vivo activity atsiRNA doses as low as 0.01 mg/kg in rodents, as well as silencing of atherapeutically significant gene (TTR) in non-human primates. Notably,the TTR silencing achieved in this work (ED₅₀˜0.3 mg/kg), represents asignificant improvement in activity relative to previous reports ofLNP-siRNA mediated silencing in non-human primates. The efficacyobserved in this study is believed to represent the highest level ofpotency observed for an RNAi therapeutic in non-human primates to date.

Accordingly, in certain embodiments, the present invention specificallyprovides for improved compositions for the delivery of siRNA molecules.It is shown herein that these compositions are effective indown-regulating the protein levels and/or mRNA levels of targetproteins. The lipid particles and compositions of the present inventionmay be used for a variety of purposes, including the delivery ofassociated or encapsulated therapeutic agents to cells, both in vitro orin vivo. Accordingly, the present invention provides methods of treatingdiseases or disorders in a subject in need thereof, by contacting thesubject with a lipid particle of the present invention associated with asuitable therapeutic agent.

As described herein, the lipid particles of the present invention areparticularly useful for the delivery of nucleic acids, including, e.g.,siRNA molecules and plasmids. Therefore, the lipid particles andcompositions of the present invention may be used to modulate theexpression of target genes and proteins both in vitro and in vivo bycontacting cells with a lipid particle of the present inventionassociated with a nucleic acid that reduces target gene expression(e.g., an siRNA) or a nucleic acid that may be used to increaseexpression of a desired protein (e.g., a plasmid encoding the desiredprotein).

Various exemplary embodiments of the cationic lipids of the presentinvention, as well as lipid particles and compositions comprising thesame, and their use to deliver therapeutic agents and modulate gene andprotein expression are described in further detail below.

A. Amino Lipids

The present invention provides novel amino lipids that areadvantageously used in lipid particles of the present invention for thein vivo delivery of therapeutic agents to cells, including amino lipidshaving the following structures.

In one embodiment of the invention, the amino lipid has the followingstructure (I):

wherein

R¹ and R² are either the same or different and independently optionallysubstituted C₁₂-C₂₄ alkyl, optionally substituted C₁₂-C₂₄ alkenyl,optionally substituted C₁₂-C₂₄ alkynyl, or optionally substitutedC₁₂-C₂₄ acyl;

R³ and R⁴ are either the same or different and independently optionallysubstituted C₁-C₆ alkyl, optionally substituted C₁-C₆ alkenyl, oroptionally substituted C₁-C₆ alkynyl or R³ and R⁴ may join to form anoptionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or2 heteroatoms chosen from nitrogen and oxygen;

R⁵ is either absent or is hydrogen or C₁-C₆ alkyl to provide aquaternary amine;

m, n, and p are either the same or different and independently either 0or 1 with the proviso that m, n, and p are not simultaneously 0;

q is 2, 3, or 4; and

Y and Z are either the same or different and independently O, S, or NH.

In one particular embodiment, q is 2.

“Alkyl” means a straight chain or branched, noncyclic or cyclic,saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms.Representative saturated straight chain alkyls include methyl, ethyl,n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturatedbranched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl,isopentyl, and the like. Representative saturated cyclic alkyls includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; whileunsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, andthe like.

“Alkenyl” means an alkyl, as defined above, containing at least onedouble bond between adjacent carbon atoms. Alkenyls include both cis andtrans isomers. Representative straight chain and branched alkenylsinclude ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl,1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,2,3-dimethyl-2-butenyl, and the like.

“Alkynyl” means any alkyl or alkenyl, as defined above, whichadditionally contains at least one triple bond between adjacent carbons.Representative straight chain and branched alkynyls include acetylenyl,propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1butynyl, and the like.

“Acyl” means any alkyl, alkenyl, or alkynyl wherein the carbon at thepoint of attachment is substituted with an oxo group, as defined below.For example, —C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl are acylgroups.

“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-memberedbicyclic, heterocyclic ring which is either saturated, unsaturated, oraromatic, and which contains from 1 or 2 heteroatoms independentlyselected from nitrogen, oxygen and sulfur, and wherein the nitrogen andsulfur heteroatoms may be optionally oxidized, and the nitrogenheteroatom may be optionally quaternized, including bicyclic rings inwhich any of the above heterocycles are fused to a benzene ring. Theheterocycle may be attached via any heteroatom or carbon atom.Heterocycles include heteroaryls as defined below. Heterocycles includemorpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl,hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

The terms “optionally substituted alkyl”, “optionally substitutedalkenyl”, “optionally substituted alkynyl”, “optionally substitutedacyl”, and “optionally substituted heterocycle” means that, whensubstituted, at least one hydrogen atom is replaced with a substituent.In the case of an oxo substituent (═O) two hydrogen atoms are replaced.In this regard, substituents include oxo, halogen, heterocycle, —CN,—OR^(x), —NR^(x)R^(y), —NR^(x)C(═O)R^(y), —NR^(x)SO₂R^(y), —C(═O)R^(x),—C(═O)OR^(x), —C(═O)NR^(x)R^(y), —SO_(n)R^(x) and —SO_(n)NR^(x)R^(y),wherein n is 0, 1 or 2, R^(x) and R^(y) are the same or different andindependently hydrogen, alkyl or heterocycle, and each of said alkyl andheterocycle substituents may be further substituted with one or more ofoxo, halogen, —OH, —CN, alkyl, —OR^(x), heterocycle, —NR^(x)R^(y),—NR^(x)C(═O)R^(y), —NR^(x)SO₂R^(y), —C(═O)R^(x), —C(═O)OR^(x),—C(═O)NR^(x)R^(y), —SO_(n)R^(x) and —SO_(n)NR^(x)R^(y).

“Halogen” means fluoro, chloro, bromo and iodo.

In certain embodiments, the amino lipid has the following structure(II):

or salts thereof, wherein

R¹ and R² are either the same or different and independently optionallysubstituted C₁₂-C₂₄ alkyl, optionally substituted C₁₂-C₂₄ alkenyl,optionally substituted C₁₂-C₂₄ alkynyl, or optionally substitutedC₁₂-C₂₄ acyl;

R³ and R⁴ are either the same or different and independently optionallysubstituted C₁-C₆ alkyl, optionally substituted C₁-C₆ alkenyl, oroptionally substituted C₁-C₆ alkynyl or R³ and R⁴ may join to form anoptionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or2 heteroatoms chosen from nitrogen and oxygen;

R⁵ is either absent or is hydrogen or C₁-C₆ alkyl to provide aquaternary amine;

m, n, and p are either the same or different and independently either 0or 1 with the proviso that m, n, and p are not simultaneously 0;

Y and Z are either the same or different and independently O, S, or NH.

In certain embodiments, the amino lipid has the following structure(III):

wherein

n is 2, 3, or 4.

In one particular embodiment, n is 2.

In certain embodiments, an amino lipid of the present invention has oneof the following structures:

In certain embodiments, an amino lipid of the present invention has oneof the following structures:

In some embodiments, the methods of the invention may require the use ofprotecting groups. Protecting group methodology is well known to thoseskilled in the art (see, for example, Protective Groups in OrganicSynthesis, Green, T. W. et. al., Wiley-Interscience, New York City,1999). Briefly, protecting groups within the context of this inventionare any group that reduces or eliminates unwanted reactivity of afunctional group. A protecting group can be added to a functional groupto mask its reactivity during certain reactions and then removed toreveal the original functional group. In some embodiments an “alcoholprotecting group” is used. An “alcohol protecting group” is any groupwhich decreases or eliminates unwanted reactivity of an alcoholfunctional group. Protecting groups can be added and removed usingtechniques well known in the art.

The compounds of the present invention may be prepared by known organicsynthesis techniques, including the methods described in more detail inthe Examples. In general, the compounds of structure (I) above may bemade by the following Reaction Schemes 1 or 2, wherein all substituentsare as defined above unless indicated otherwise.

Compounds of structure (I) wherein m is 1 and p is 0 can be preparedaccording to Reaction Scheme 1. Ketone 1 and Grignard reagent 2, whereinP is an alcohol protecting group such as trityl, can be purchased orprepared according to methods known to those of ordinary skill in theart. Reaction of 1 and 2 yields alcohol 3. Deprotection of 3, forexample by treatment with mild acid, followed by bromination with anappropriate bromination reagent, for example phosphorous tribromide,yields 4 and 5 respectively. Treatment of bromide 5 with 6 yields theheterocyclic compound 7. Treatment of 7 with amine 8 then yields acompound of structure (I) wherein m is 1 and R⁵ is absent (9). Furthertreatment of 9 with chloride 10 yields compounds of structure (I)wherein m is 1 and R⁵ is present.

Compounds of structure (I) wherein m and p are 0 can be preparedaccording to Reaction Scheme 2. Ketone 1 and bromide 6 can be purchasedor prepared according to methods known to those of ordinary skill in theart. Reaction of 1 and 6 yields heterocycle 12. Treatment of 12 withamine 8 yields compounds of structure (I) wherein m is 0 and R⁵ isabsent (13). Further treatment of 13 with 10 produces compounds ofstructure (I) wherein w is 0 and R⁵ is present.

In certain embodiments where m and p are 1 and n is 0, compounds of thisinvention can be prepared according to Reaction Scheme 3. Compounds 12and 13 can be purchased or prepared according to methods know to thoseof ordinary skill in the art. Reaction of 12 and 13 yields a compound ofstructure (I) where R⁵ is absent (14). In other embodiments where R⁵ ispresent, 13 can be treated with 10 to obtain compounds of structure 15.

In certain other embodiments where either m or p is 1 and n is 0,compounds of this invention can be prepared according to Reaction Scheme4. Compound 16 can be purchased or prepared according to methods know tothose of ordinary skill in the art and reacted with 13 to yield acompound of structure (I) where R⁵ is absent (17). Other embodiments ofstructure (I) where R⁵ is present can be prepared by treatment of 17with 10 to yield compounds of structure 18.

In certain specific embodiments of structure (I) where n is 1 and m andp are 0, compounds of this invention can be prepared according toReaction Scheme 5. Compound 19 can be purchased or prepared according tomethods known to those of ordinary skill in the art. Reaction of 19 withformaldehyde followed by removal of an optional alcohol protecting group(P), yields alcohol 20. Bromination of 20 followed by treatment withamine 8 yields 22. Compound 22 can then be treated with n-butyl lithiumand R¹I followed by further treatment with n-butyl lithium and R²I toyield a compound of structure (I) where R⁵ is absent (23). Furthertreatment of 23 with 10 yields a compound of structure (I) where R⁵ ispresent (24).

In particular embodiments, the amino lipids of the present invention arecationic lipids. As used herein, the term “amino lipid” is meant toinclude those lipids having one or two fatty acid or fatty alkyl chainsand an amino head group (including an alkylamino or dialkylamino group)that may be protonated to form a cationic lipid at physiological pH.

Other amino lipids would include those having alternative fatty acidgroups and other dialkylamino groups, including those in which the alkylsubstituents are different (e.g., N-ethyl-N-methylamino-,N-propyl-N-ethylamino- and the like). For those embodiments in which R¹¹and R¹² are both long chain alkyl or acyl groups, they can be the sameor different. In general, amino lipids having less saturated acyl chainsare more easily sized, particularly when the complexes must be sizedbelow about 0.3 microns, for purposes of filter sterilization. Aminolipids containing unsaturated fatty acids with carbon chain lengths inthe range of C₁₄ to C₂₂ are preferred. Other scaffolds can also be usedto separate the amino group and the fatty acid or fatty alkyl portion ofthe amino lipid. Suitable scaffolds are known to those of skill in theart.

In certain embodiments, amino or cationic lipids of the presentinvention have at least one protonatable or deprotonatable group, suchthat the lipid is positively charged at a pH at or below physiologicalpH (e.g. pH 7.4), and neutral at a second pH, preferably at or abovephysiological pH. It will, of course, be understood that the addition orremoval of protons as a function of pH is an equilibrium process, andthat the reference to a charged or a neutral lipid refers to the natureof the predominant species and does not require that all of the lipid bepresent in the charged or neutral form. Lipids that have more than oneprotonatable or deprotonatable group, or which are zwiterrionic, are notexcluded from use in the invention.

In certain embodiments, protonatable lipids according to the inventionhave a pKa of the protonatable group in the range of about 4 to about11. Most preferred is pKa of about 4 to about 7, because these lipidswill be cationic at a lower pH formulation stage, while particles willbe largely (though not completely) surface neutralized at physiologicalpH around pH 7.4. One of the benefits of this pKa is that at least somenucleic acid associated with the outside surface of the particle willlose its electrostatic interaction at physiological pH and be removed bysimple dialysis; thus greatly reducing the particle's susceptibility toclearance.

B. Lipid Particles

The present invention also provides lipid particles comprising one ormore of the amino lipids described above. Lipid particles include, butare not limited to, liposomes. As used herein, a liposome is a structurehaving lipid-containing membranes enclosing an aqueous interior.Liposomes may have one or more lipid membranes. The inventioncontemplates both single-layered liposomes, which are referred to asunilamellar, and multi-layered liposomes, which are referred to asmultilamellar. When complexed with nucleic acids, lipid particles mayalso be lipoplexes, which are composed of cationic lipid bilayerssandwiched between DNA layers, as described, e.g., in Felgner,Scientific American.

The lipid particles of the present invention may further comprise one ormore additional lipids and/or other components, such as cholesterol.Other lipids may be included in the liposome compositions of the presentinvention for a variety of purposes, such as to prevent lipid oxidationor to attach ligands onto the liposome surface. Any of a number oflipids may be present in liposomes of the present invention, includingamphipathic, neutral, cationic, and anionic lipids. Such lipids can beused alone or in combination. Specific examples of additional lipidcomponents that may be present are described below.

In certain embodiments, lipid particles of the present inventioncomprise an amino lipid described above, a non-cationic or neutrallipid, and a conjugated lipid that inhibits particle aggregation. Incertain embodiments, lipid particles of the present invention comprisean amino lipid described above, a non-cationic or neutral lipid, asterol, and a conjugated lipid that inhibits particle aggregation. Inparticular embodiments, these lipid particles further comprise acationic lipid in addition to the amino lipid of the present invention.

Additional components that may be present in a lipid particle of thepresent invention include bilayer stabilizing components such aspolyamide oligomers (see, e.g., U.S. Pat. No. 6,320,017), peptides,proteins, detergents, lipid-derivatives, such as PEG coupled tophosphatidylethanolamine and PEG conjugated to ceramides (see, U.S. Pat.No. 5,885,613).

Examples of lipids that reduce aggregation of particles during formationinclude polyethylene glycol (PEG)-modified lipids, monosialogangliosideGm1, and polyamide oligomers (“PAO”) such as (described in U.S. Pat. No.6,320,017). Other compounds with uncharged, hydrophilic, steric-barriermoieties, which prevent aggregation during formulation, like PEG, Gm1 orATTA, can also be coupled to lipids for use as in the methods andcompositions of the invention. ATTA-lipids are described, e.g., in U.S.Pat. No. 6,320,017, and PEG-lipid conjugates are described, e.g., inU.S. Pat. Nos. 5,820,873, 5,534,499 and 5,885,613. Typically, theconcentration of the lipid component selected to reduce aggregation isabout 1 to 15% (by mole percent of lipids).

Specific examples of PEG-modified lipids (or lipid-polyoxyethyleneconjugates) that are useful in the present invention can have a varietyof “anchoring” lipid portions to secure the PEG portion to the surfaceof the lipid vesicle. Examples of suitable PEG-modified lipids includePEG-modified phosphatidylethanolamine and phosphatidic acid,PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20) which aredescribed in co-pending U.S. Ser. No. 08/486,214, incorporated herein byreference, PEG-modified dialkylamines and PEG-modified1,2-diacyloxypropan-3-amines. Particularly preferred are PEG-modifieddiacylglycerols and dialkylglycerols.

In particular embodiments, a PEG-lipid is selected from:

In embodiments where a sterically-large moiety such as PEG or ATTA areconjugated to a lipid anchor, the selection of the lipid anchor dependson what type of association the conjugate is to have with the lipidparticle. It is well known that mePEG(mw2000)-diastearoylphosphatidylethanolamine (PEG-DSPE) will remainassociated with a liposome until the particle is cleared from thecirculation, possibly a matter of days. Other conjugates, such asPEG-CerC20 have similar staying capacity. PEG-CerC14, however, rapidlyexchanges out of the formulation upon exposure to serum, with a T_(1/2)less than 60 mins. in some assays. As illustrated in U.S. patentapplication Ser. No. 08/486,214, at least three characteristicsinfluence the rate of exchange: length of acyl chain, saturation of acylchain, and size of the steric-barrier head group. Compounds havingsuitable variations of these features may be useful for the invention.For some therapeutic applications it may be preferable for thePEG-modified lipid to be rapidly lost from the nucleic acid-lipidparticle in vivo and hence the PEG-modified lipid will possessrelatively short lipid anchors. In other therapeutic applications it maybe preferable for the nucleic acid-lipid particle to exhibit a longerplasma circulation lifetime and hence the PEG-modified lipid willpossess relatively longer lipid anchors.

It should be noted that aggregation preventing compounds do notnecessarily require lipid conjugation to function properly. Free PEG orfree ATTA in solution may be sufficient to prevent aggregation. If theparticles are stable after formulation, the PEG or ATTA can be dialyzedaway before administration to a subject.

The term “non-cationic lipid” refers to any amphipathic lipid as well asany other neutral lipid or anionic lipid. Non-cationic lipids used inthe lipid particles, e.g., SNALP, of the present invention can be any ofa variety of neutral uncharged, zwitterionic, or anionic lipids capableof producing a stable complex.

The term “neutral lipid” refers to any of a number of lipid species thatexist either in an uncharged or neutral zwitterionic form at a selectedpH. At physiological pH, such lipids include, for example,diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.

The term “anionic lipid” refers to any lipid that is negatively chargedat physiological pH. These lipids include, but are not limited to,phosphatidylglycerols, cardiolipins, diacylphosphatidylserines,diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines,N-succinyl phosphatidylethanolamines,N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols,palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifyinggroups joined to neutral lipids. Such lipids include, for examplediacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. Theselection of neutral lipids for use in the particles described herein isgenerally guided by consideration of, e.g., liposome size and stabilityof the liposomes in the bloodstream. Preferably, the neutral lipidcomponent is a lipid having two acyl groups, (i.e.,diacylphosphatidylcholine and diacylphosphatidylethanolamine). Lipidshaving a variety of acyl chain groups of varying chain length and degreeof saturation are available or may be isolated or synthesized bywell-known techniques. In one group of embodiments, lipids containingsaturated fatty acids with carbon chain lengths in the range of C₁₄ toC₂₂ are preferred. In another group of embodiments, lipids with mono ordiunsaturated fatty acids with carbon chain lengths in the range of C₁₄to C₂₂ are used. Additionally, lipids having mixtures of saturated andunsaturated fatty acid chains can be used. Preferably, the neutrallipids used in the present invention are DOPE, DSPC, POPC, or anyrelated phosphatidylcholine. The neutral lipids useful in the presentinvention may also be composed of sphingomyelin, dihydrosphingomyeline,or phospholipids with other head groups, such as serine and inositol.

Non-limiting examples of non-cationic lipids include phospholipids suchas lecithin, phosphatidylethanolamine, lysolecithin,lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin,phosphatidic acid, cerebrosides, dicetylphosphate,distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoyl-phosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),palmitoyloleyolphosphatidylglycerol (POPG),dioleoylphosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),dipalmitoyl-phosphatidylethanolamine (DPPE),dimyristoyl-phosphatidylethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE),monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine,dielaidoyl-phosphatidylethanolamine (DEPE),stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine,dilinoleoylphosphatidylcholine, and mixtures thereof. Otherdiacylphosphatidylcholine and diacylphosphatidylethanolaminephospholipids can also be used. The acyl groups in these lipids arepreferably acyl groups derived from fatty acids having C₁₀-C₂₄ carbonchains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

Additional examples of non-cationic lipids include sterols such ascholesterol and derivatives thereof such as cholestanol, cholestanone,cholestenone, and coprostanol.

In some embodiments, the non-cationic lipid present in the lipidparticle, e.g., SNALP, comprises or consists of cholesterol, e.g., aphospholipid-free SNALP. In other embodiments, the non-cationic lipidpresent in the lipid particle, e.g., SNALP comprises or consists of oneor more phospholipids, e.g., a cholesterol-free SNALP. In furtherembodiments, the non-cationic lipid present in the SNALP comprises orconsists of a mixture of one or more phospholipids and cholesterol.

Other examples of non-cationic lipids suitable for use in the presentinvention include nonphosphorous containing lipids such as, e.g.,stearylamine, dodecylamine, hexadecylamine, acetyl palmitate,glycerolricinoleate, hexadecyl stereate, isopropyl myristate, amphotericacrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfatepolyethyloxylated fatty acid amides, dioctadecyldimethyl ammoniumbromide, ceramide, sphingomyelin, and the like.

The non-cationic lipid typically comprises from about 13 mol % to about49.5 mol %, about 20 mol % to about 45 mol %, about 25 mol % to about 45mol %, about 30 mol % to about 45 mol %, about 35 mol % to about 45 mol%, about 20 mol % to about 40 mol %, about 25 mol % to about 40 mol %,or about 30 mol % to about 40 mol % of the total lipid present in theparticle.

The sterol component of the lipid mixture, when present, can be any ofthose sterols conventionally used in the field of liposome, lipidvesicle or lipid particle preparation. A preferred sterol ischolesterol.

Other cationic lipids, which carry a net positive charge at aboutphysiological pH, in addition to those specifically described above, mayalso be included in lipid particles of the present invention. Suchcationic lipids include, but are not limited to,N,N-dioleyl-N,N-dimethylammonium chloride (“DODAC”);N-(2,3-dioleyloxy)propyl-N,N-N-triethylammonium chloride (“DOTMA”);N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”);N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTAP”);1,2-Dioleyloxy-3-trimethylaminopropane chloride salt (“DOTAP.Cl”);3β-(N-(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (“DC-Chol”),N-(1-(2,3-dioleyloxy)propyl)-N′-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoracetate (“DOSPA”), dioctadecylamidoglycyl carboxyspermine(“DOGS”), 1,2-dileoyl-sn-3-phosphoethanolamine (“DOPE”),1,2-dioleoyl-3-dimethylammonium propane (“DODAP”),N,N-dimethyl-2,3-dioleyloxy)propylamine (“DODMA”), andN-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (“DMRIE”). Additionally, a number of commercial preparations ofcationic lipids can be used, such as, e.g., LIPOFECTIN (including DOTMAand DOPE, available from GIBCO/BRL), and LIPOFECTAMINE (comprising DOSPAand DOPE, available from GIBCO/BRL). In particular embodiments, acationic lipid is an amino lipid.

In numerous embodiments, amphipathic lipids are included in lipidparticles of the present invention. “Amphipathic lipids” refer to anysuitable material, wherein the hydrophobic portion of the lipid materialorients into a hydrophobic phase, while the hydrophilic portion orientstoward the aqueous phase. Such compounds include, but are not limitedto, phospholipids, aminolipids, and sphingolipids. Representativephospholipids include sphingomyelin, phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,phosphatidic acid, palmitoyloleoyl phosphatidylcholine,lysophosphatidylcholine, lysophosphatidylethanolamine,dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine,distearoylphosphatidylcholine, or dilinoleoylphosphatidylcholine. Otherphosphorus-lacking compounds, such as sphingolipids, glycosphingolipidfamilies, diacylglycerols, and β-acyloxyacids, can also be used.Additionally, such amphipathic lipids can be readily mixed with otherlipids, such as triglycerides and sterols.

Also suitable for inclusion in the lipid particles of the presentinvention are programmable fusion lipids. Such lipid particles havelittle tendency to fuse with cell membranes and deliver their payloaduntil a given signal event occurs. This allows the lipid particle todistribute more evenly after injection into an organism or disease sitebefore it starts fusing with cells. The signal event can be, forexample, a change in pH, temperature, ionic environment, or time. In thelatter case, a fusion delaying or “cloaking” component, such as anATTA-lipid conjugate or a PEG-lipid conjugate, can simply exchange outof the lipid particle membrane over time. By the time the lipid particleis suitably distributed in the body, it has lost sufficient cloakingagent so as to be fusogenic. With other signal events, it is desirableto choose a signal that is associated with the disease site or targetcell, such as increased temperature at a site of inflammation.

In certain embodiments, it is desirable to target the lipid particles ofthis invention using targeting moieties that are specific to a cell typeor tissue. Targeting of lipid particles using a variety of targetingmoieties, such as ligands, cell surface receptors, glycoproteins,vitamins (e.g., riboflavin) and monoclonal antibodies, has beenpreviously described (see, e.g., U.S. Pat. Nos. 4,957,773 and4,603,044). The targeting moieties can comprise the entire protein orfragments thereof. Targeting mechanisms generally require that thetargeting agents be positioned on the surface of the lipid particle insuch a manner that the target moiety is available for interaction withthe target, for example, a cell surface receptor. A variety of differenttargeting agents and methods are known and available in the art,including those described, e.g., in Sapra, P. and Allen, T M, Prog.Lipid Res. 42(5):439-62 (2003); and Abra, R M et al., J. Liposome Res.12:1-3, (2002).

The use of lipid particles, i.e., liposomes, with a surface coating ofhydrophilic polymer chains, such as polyethylene glycol (PEG) chains,for targeting has been proposed (Allen, et al., Biochimica et BiophysicaActa 1237: 99-108 (1995); DeFrees, et al., Journal of the AmericanChemistry Society 118: 6101-6104 (1996); Blume, et al., Biochimica etBiophysica Acta 1149: 180-184 (1993); Klibanov, et al., Journal ofLiposome Research 2: 321-334 (1992); U.S. Pat. No. 5,013,556; Zalipsky,Bioconjugate Chemistry 4: 296-299 (1993); Zalipsky, FEBS Letters 353:71-74 (1994); Zalipsky, in Stealth Liposomes Chapter 9 (Lasic andMartin, Eds) CRC Press, Boca Raton, Fla. (1995). In one approach, aligand, such as an antibody, for targeting the lipid particle is linkedto the polar head group of lipids forming the lipid particle. In anotherapproach, the targeting ligand is attached to the distal ends of the PEGchains forming the hydrophilic polymer coating (Klibanov, et al.,Journal of Liposome Research 2: 321-334 (1992); Kirpotin et al., FEBSLetters 388: 115-118 (1996)).

Standard methods for coupling the target agents can be used. Forexample, phosphatidylethanolamine, which can be activated for attachmentof target agents, or derivatized lipophilic compounds, such aslipid-derivatized bleomycin, can be used. Antibody-targeted liposomescan be constructed using, for instance, liposomes that incorporateprotein A (see, Renneisen, et al., J. Bio. Chem., 265:16337-16342 (1990)and Leonetti, et al., Proc. Natl. Acad. Sci. (USA), 87:2448-2451 (1990).Other examples of antibody conjugation are disclosed in U.S. Pat. No.6,027,726, the teachings of which are incorporated herein by reference.Examples of targeting moieties can also include other proteins, specificto cellular components, including antigens associated with neoplasms ortumors. Proteins used as targeting moieties can be attached to theliposomes via covalent bonds (see, Heath, Covalent Attachment ofProteins to Liposomes, 149 Methods in Enzymology 111-119 (AcademicPress, Inc. 1987)). Other targeting methods include the biotin-avidinsystem.

In one exemplary embodiment, the lipid particle comprises a mixture ofan amino lipid of the present invention, neutral lipids (other than anamino lipid), a sterol (e.g., cholesterol) and a PEG-modified lipid(e.g., a PEG-S-DMG, PEG-C-DOMG or PEG-DMA). In certain embodiments, thelipid mixture consists of or consists essentially of an amino lipid ofthe present invention, a neutral lipid, cholesterol, and a PEG-modifiedlipid. In further preferred embodiments, the lipid particle consists ofor consists essentially of the above lipid mixture in molar ratios ofabout 20-70% amino lipid: 5-45% neutral lipid:20-55% cholesterol:0.5-15%PEG-modified lipid.

In particular embodiments, the lipid particle consists of or consistsessentially of DLin-K-C2-DMA, DSPC, Chol, and either PEG-S-DMG,PEG-C-DOMG or PEG-DMA, e.g., in a molar ratio of about 20-60%DLin-K-C2-DMA: 5-25% DSPC:25-55% Chol:0.5-15% PEG-S-DMG, PEG-C-DOMG orPEG-DMA. In particular embodiments, the molar lipid ratio isapproximately 40/10/40/10 (mol % DLin-K-C2-DMA/DSPC/Chol/PEG-S-DMG orDLin-K-C2-DMA/DSPC/Chol/PEG-C-DOMG or DLin-K-C2-DMA/DSPC/Chol/PEG-DMA)or 35/15/40/10 mol % DLin-K-C2-DMA/DSPC/Chol/PEG-S-DMG orDLin-K-C2-DMA/DSPC/Chol/PEG-C-DOMG or DLin-K-C2-DMA/DSPC/Chol/PEG-DMA.In another group of embodiments, the neutral lipid in these compositionsis replaced with POPC, DOPE or SM.

In particular embodiments, the lipid particle consists of or consistsessentially of DLin-K²-DMA, DSPC, Chol, and either PEG-S-DMG, PEG-C-DOMGor PEG-DMA, e.g., in a molar ratio of about 20-60% DLin-K²-DMA: 5-25%DSPC:25-55% Chol:0.5-15% PEG-S-DMG, PEG-C-DOMG or PEG-DMA. In particularembodiments, the molar lipid ratio is approximately 40/10/40/10 (mol %DLin-K²-DMA/DSPC/Chol/PEG-S-DMG or DLin-K²-DMA/DSPC/Chol/PEG-C-DOMG orDLin-K²-DMA/DSPC/Chol/PEG-DMA) or 35/15/40/10 mol %DLin-K²-DMA/DSPC/Chol/PEG-S-DMG or DLin-K²-DMA/DSPC/Chol/PEG-C-DOMG orDLin-K²-DMA/DSPC/Chol/PEG-DMA. In another group of embodiments, theneutral lipid in these compositions is replaced with POPC, DOPE or SM.

In particular embodiments, the lipid particle consists of or consistsessentially of DLin-K6-DMA, DSPC, Chol, and either PEG-S-DMG, PEG-C-DOMGor PEG-DMA, e.g., in a molar ratio of about 20-60% DLin-K6-DMA: 5-25%DSPC:25-55% Chol:0.5-15% PEG-S-DMG, PEG-C-DOMG or PEG-DMA. In particularembodiments, the molar lipid ratio is approximately 40/10/40/10 (mol %DLin-K6-DMA/DSPC/Chol/PEG-S-DMG or DLin-K6-DMA/DSPC/Chol/PEG-C-DOMG orDLin-K6-DMA/DSPC/Chol/PEG-DMA) or 35/15/40/10 mol %DLin-K6-DMA/DSPC/Chol/PEG-S-DMG or DLin-K6-DMA/DSPC/Chol/PEG-C-DOMG orDLin-K6-DMA/DSPC/Chol/PEG-DMA. In another group of embodiments, theneutral lipid in these compositions is replaced with POPC, DOPE or SM.

C. Therapeutic Agent-Lipid Particle Compositions and Formulations

The present invention includes compositions comprising a lipid particleof the present invention and an active agent, wherein the active agentis associated with the lipid particle. In particular embodiments, theactive agent is a therapeutic agent. In particular embodiments, theactive agent is encapsulated within an aqueous interior of the lipidparticle. In other embodiments, the active agent is present within oneor more lipid layers of the lipid particle. In other embodiments, theactive agent is bound to the exterior or interior lipid surface of alipid particle.

“Fully encapsulated” as used herein indicates that the nucleic acid inthe particles is not significantly degraded after exposure to serum or anuclease assay that would significantly degrade the free nucleic acid.In a fully encapsulated system, preferably less than 25% of particlenucleic acid is degraded in a treatment that would normally degrade 100%of free nucleic acid, more preferably less than 10% and most preferablyless than 5% of the particle nucleic acid is degraded. Alternatively,full encapsulation may be determined by an Oligreen® assay. Oligreen® isan ultra-sensitive fluorescent nucleic acid stain for quantitatingoligonucleotides and single-stranded DNA or RNA in solution (availablefrom Invitrogen Corporation, Carlsbad, Calif.). Fully encapsulated alsosuggests that the particles are serum stable, that is, that they do notrapidly decompose into their component parts upon in vivoadministration.

Active agents, as used herein, include any molecule or compound capableof exerting a desired effect on a cell, tissue, organ, or subject. Sucheffects may be biological, physiological, or cosmetic, for example.Active agents may be any type of molecule or compound, including e.g.,nucleic acids, peptides and polypeptides, including, e.g., antibodies,such as, e.g., polyclonal antibodies, monoclonal antibodies, antibodyfragments; humanized antibodies, recombinant antibodies, recombinanthuman antibodies, and Primatized™ antibodies, cytokines, growth factors,apoptotic factors, differentiation-inducing factors, cell surfacereceptors and their ligands; hormones; and small molecules, includingsmall organic molecules or compounds.

In one embodiment, the active agent is a therapeutic agent, or a salt orderivative thereof. Therapeutic agent derivatives may be therapeuticallyactive themselves or they may be prodrugs, which become active uponfurther modification. Thus, in one embodiment, a therapeutic agentderivative retains some or all of the therapeutic activity as comparedto the unmodified agent, while in another embodiment, a therapeuticagent derivative lacks therapeutic activity.

In various embodiments, therapeutic agents include any therapeuticallyeffective agent or drug, such as anti-inflammatory compounds,anti-depressants, stimulants, analgesics, antibiotics, birth controlmedication, antipyretics, vasodilators, anti-angiogenics, cytovascularagents, signal transduction inhibitors, cardiovascular drugs, e.g.,anti-arrhythmic agents, vasoconstrictors, hormones, and steroids.

In certain embodiments, the therapeutic agent is an oncology drug, whichmay also be referred to as an anti-tumor drug, an anti-cancer drug, atumor drug, an antineoplastic agent, or the like. Examples of oncologydrugs that may be used according to the invention include, but are notlimited to, adriamycin, alkeran, allopurinol, altretamine, amifostine,anastrozole, araC, arsenic trioxide, azathioprine, bexarotene, biCNU,bleomycin, busulfan intravenous, busulfan oral, capecitabine (Xeloda),carboplatin, carmustine, CCNU, celecoxib, chlorambucil, cisplatin,cladribine, cyclosporin A, cytarabine, cytosine arabinoside,daunorubicin, cytoxan, daunorubicin, dexamethasone, dexrazoxane,dodetaxel, doxorubicin, doxorubicin, DTIC, epirubicin, estramustine,etoposide phosphate, etoposide and VP-16, exemestane, FK506,fludarabine, fluorouracil, 5-FU, gemcitabine (Gemzar),gemtuzumab-ozogamicin, goserelin acetate, hydrea, hydroxyurea,idarubicin, ifosfamide, imatinib mesylate, interferon, irinotecan(Camptostar, CPT-111), letrozole, leucovorin, leustatin, leuprolide,levamisole, litertinoin, megastrol, melphalan, L-PAM, mesna,methotrexate, methoxsalen, mithramycin, mitomycin, mitoxantrone,nitrogen mustard, paclitaxel, pamidronate, Pegademase, pentostatin,porfimer sodium, prednisone, rituxan, streptozocin, STI-571, tamoxifen,taxotere, temozolamide, teniposide, VM-26, topotecan (Hycamtin),toremifene, tretinoin, ATRA, valrubicin, velban, vinblastine,vincristine, VP16, and vinorelbine. Other examples of oncology drugsthat may be used according to the invention are ellipticin andellipticin analogs or derivatives, epothilones, intracellular kinaseinhibitors and camptothecins.

1. Nucleic Acid-Lipid Particles

In certain embodiments, lipid particles of the present invention areassociated with a nucleic acid, resulting in a nucleic acid-lipidparticle. In particular embodiments, the nucleic acid is partially orfully encapsulated in the lipid particle. As used herein, the term“nucleic acid” is meant to include any oligonucleotide orpolynucleotide. Fragments containing up to 50 nucleotides are generallytermed oligonucleotides, and longer fragments are calledpolynucleotides. In particular embodiments, oligonucleotides of thepresent invention are 20-50 nucleotides in length.

In the context of this invention, the terms “polynucleotide” and“oligonucleotide” refer to a polymer or oligomer of nucleotide ornucleoside monomers consisting of naturally occurring bases, sugars andintersugar (backbone) linkages. The terms “polynucleotide” and“oligonucleotide” also includes polymers or oligomers comprisingnon-naturally occurring monomers, or portions thereof, which functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of properties such as, for example,enhanced cellular uptake and increased stability in the presence ofnucleases.

Oligonucleotides are classified as deoxyribooligonucleotides orribooligonucleotides. A deoxyribooligonucleotide consists of a 5-carbonsugar called deoxyribose joined covalently to phosphate at the 5′ and 3′carbons of this sugar to form an alternating, unbranched polymer. Aribooligonucleotide consists of a similar repeating structure where the5-carbon sugar is ribose.

The nucleic acid that is present in a lipid-nucleic acid particleaccording to this invention includes any form of nucleic acid that isknown. The nucleic acids used herein can be single-stranded DNA or RNA,or double-stranded DNA or RNA, or DNA-RNA hybrids. Examples ofdouble-stranded DNA include structural genes, genes including controland termination regions, and self-replicating systems such as viral orplasmid DNA. Examples of double-stranded RNA include siRNA and other RNAinterference reagents. Single-stranded nucleic acids include, e.g.,antisense oligonucleotides, ribozymes, microRNA, and triplex-formingoligonucleotides.

Nucleic acids of the present invention may be of various lengths,generally dependent upon the particular form of nucleic acid. Forexample, in particular embodiments, plasmids or genes may be from about1,000 to 100,000 nucleotide residues in length. In particularembodiments, oligonucleotides may range from about 10 to 100 nucleotidesin length. In various related embodiments, oligonucleotides, bothsingle-stranded, double-stranded, and triple-stranded, may range inlength from about 10 to about 50 nucleotides, from about 20 to about 50nucleotides, from about 15 to about 30 nucleotides, from about 20 toabout 30 nucleotides in length.

In particular embodiments, an oligonucleotide (or a strand thereof) ofthe present invention specifically hybridizes to or is complementary toa target polynucleotide. “Specifically hybridizable” and “complementary”are terms which are used to indicate a sufficient degree ofcomplementarity such that stable and specific binding occurs between theDNA or RNA target and the oligonucleotide. It is understood that anoligonucleotide need not be 100% complementary to its target nucleicacid sequence to be specifically hybridizable. An oligonucleotide isspecifically hybridizable when binding of the oligonucleotide to thetarget interferes with the normal function of the target molecule tocause a loss of utility or expression therefrom, and there is asufficient degree of complementarity to avoid non-specific binding ofthe oligonucleotide to non-target sequences under conditions in whichspecific binding is desired, i.e., under physiological conditions in thecase of in vivo assays or therapeutic treatment, or, in the case of invitro assays, under conditions in which the assays are conducted. Thus,in other embodiments, this oligonucleotide includes 1, 2, or 3 basesubstitutions as compared to the region of a gene or mRNA sequence thatit is targeting or to which it specifically hybridizes.

RNA Interference Nucleic Acids

In particular embodiments, nucleic acid-lipid particles of the presentinvention are associated with RNA interference (RNAi) molecules. RNAinterference methods using RNAi molecules may be used to disrupt theexpression of a gene or polynucleotide of interest. In the last 5 yearssmall interfering RNA (siRNA) has essentially replaced antisense ODN andribozymes as the next generation of targeted oligonucleotide drugs underdevelopment. SiRNAs are RNA duplexes normally 21-30 nucleotides longthat can associate with a cytoplasmic multi-protein complex known asRNAi-induced silencing complex (RISC). RISC loaded with siRNA mediatesthe degradation of homologous mRNA transcripts, therefore siRNA can bedesigned to knock down protein expression with high specificity. Unlikeother antisense technologies, siRNA function through a natural mechanismevolved to control gene expression through non-coding RNA. This isgenerally considered to be the reason why their activity is more potentin vitro and in vivo than either antisense ODN or ribozymes. A varietyof RNAi reagents, including siRNAs targeting clinically relevanttargets, are currently under pharmaceutical development, as described,e.g., in de Fougerolles, A. et al., Nature Reviews 6:443-453 (2007).

While the first described RNAi molecules were RNA:RNA hybrids comprisingboth an RNA sense and an RNA antisense strand, it has now beendemonstrated that DNA sense:RNA antisense hybrids, RNA sense:DNAantisense hybrids, and DNA:DNA hybrids are capable of mediating RNAi(Lamberton, J. S, and Christian, A. T., (2003) Molecular Biotechnology24:111-119). Thus, the invention includes the use of RNAi moleculescomprising any of these different types of double-stranded molecules. Inaddition, it is understood that RNAi molecules may be used andintroduced to cells in a variety of forms. Accordingly, as used herein,RNAi molecules encompasses any and all molecules capable of inducing anRNAi response in cells, including, but not limited to, double-strandedpolynucleotides comprising two separate strands, i.e. a sense strand andan antisense strand, e.g., small interfering RNA (siRNA);polynucleotides comprising a hairpin loop of complementary sequences,which forms a double-stranded region, e.g., shRNAi molecules, andexpression vectors that express one or more polynucleotides capable offorming a double-stranded polynucleotide alone or in combination withanother polynucleotide.

RNA interference (RNAi) may be used to specifically inhibit expressionof target polynucleotides. Double-stranded RNA-mediated suppression ofgene and nucleic acid expression may be accomplished according to theinvention by introducing dsRNA, siRNA or shRNA into cells or organisms.SiRNA may be double-stranded RNA, or a hybrid molecule comprising bothRNA and DNA, e.g., one RNA strand and one DNA strand. It has beendemonstrated that the direct introduction of siRNAs to a cell cantrigger RNAi in mammalian cells (Elshabir, S. M., et al. Nature411:494-498 (2001)). Furthermore, suppression in mammalian cellsoccurred at the RNA level and was specific for the targeted genes, witha strong correlation between RNA and protein suppression (Caplen, N. etal., Proc. Natl. Acad. Sci. USA 98:9746-9747 (2001)). In addition, itwas shown that a wide variety of cell lines, including HeLa S3, COS7,293, NIH/3T3, A549, HT-29, CHO-KI and MCF-7 cells, are susceptible tosome level of siRNA silencing (Brown, D. et al. TechNotes 9(1):1-7,available at http://www.dot.ambion.dot.com/techlib/tn/91/912.html (Sep.1, 2002)).

RNAi molecules targeting specific polynucleotides can be readilyprepared according to procedures known in the art. Structuralcharacteristics of effective siRNA molecules have been identified.Elshabir, S. M. et al. (2001) Nature 411:494-498 and Elshabir, S. M. etal. (2001), EMBO 20:6877-6888. Accordingly, one of skill in the artwould understand that a wide variety of different siRNA molecules may beused to target a specific gene or transcript. In certain embodiments,siRNA molecules according to the invention are double-stranded and 16-30or 18-25 nucleotides in length, including each integer in between. Inone embodiment, an siRNA is 21 nucleotides in length. In certainembodiments, siRNAs have 0-7 nucleotide 3′ overhangs or 0-4 nucleotide5′ overhangs. In one embodiment, an siRNA molecule has a two nucleotide3′ overhang. In one embodiment, an siRNA is 21 nucleotides in lengthwith two nucleotide 3′ overhangs (i.e. they contain a 19 nucleotidecomplementary region between the sense and antisense strands). Incertain embodiments, the overhangs are UU or dTdT 3′ overhangs.

Generally, siRNA molecules are completely complementary to one strand ofa target DNA molecule, since even single base pair mismatches have beenshown to reduce silencing. In other embodiments, siRNAs may have amodified backbone composition, such as, for example, 2′-deoxy- or2′-O-methyl modifications. However, in preferred embodiments, the entirestrand of the siRNA is not made with either 2′ deoxy or 2′-O-modifiedbases.

In one embodiment, siRNA target sites are selected by scanning thetarget mRNA transcript sequence for the occurrence of AA dinucleotidesequences. Each AA dinucleotide sequence in combination with the3′adjacent approximately 19 nucleotides are potential siRNA targetsites. In one embodiment, siRNA target sites are preferentially notlocated within the 5′ and 3′ untranslated regions (UTRs) or regions nearthe start codon (within approximately 75 bases), since proteins thatbind regulatory regions may interfere with the binding of the siRNPendonuclease complex (Elshabir, S. et al. Nature 411:494-498 (2001);Elshabir, S. et al. EMBO J. 20:6877-6888 (2001)). In addition, potentialtarget sites may be compared to an appropriate genome database, such asBLASTN 2.0.5, available on the NCBI server at www.ncbi.nlm, andpotential target sequences with significant homology to other codingsequences eliminated.

In particular embodiments, short hairpin RNAs constitute the nucleicacid component of nucleic acid-lipid particles of the present invention.Short Hairpin RNA (shRNA) is a form of hairpin RNA capable ofsequence-specifically reducing expression of a target gene. Shorthairpin RNAs may offer an advantage over siRNAs in suppressing geneexpression, as they are generally more stable and less susceptible todegradation in the cellular environment. It has been established thatsuch short hairpin RNA-mediated gene silencing works in a variety ofnormal and cancer cell lines, and in mammalian cells, including mouseand human cells. Paddison, P. et al., Genes Dev. 16(8):948-58 (2002).Furthermore, transgenic cell lines bearing chromosomal genes that codefor engineered shRNAs have been generated. These cells are able toconstitutively synthesize shRNAs, thereby facilitating long-lasting orconstitutive gene silencing that may be passed on to progeny cells.Paddison, P. et al., Proc. Natl. Acad. Sci. USA 99(3):1443-1448 (2002).

ShRNAs contain a stem loop structure. In certain embodiments, they maycontain variable stem lengths, typically from 19 to 29 nucleotides inlength, or any number in between. In certain embodiments, hairpinscontain 19 to 21 nucleotide stems, while in other embodiments, hairpinscontain 27 to 29 nucleotide stems. In certain embodiments, loop size isbetween 4 to 23 nucleotides in length, although the loop size may belarger than 23 nucleotides without significantly affecting silencingactivity. ShRNA molecules may contain mismatches, for example G-Umismatches between the two strands of the shRNA stem without decreasingpotency. In fact, in certain embodiments, shRNAs are designed to includeone or several G-U pairings in the hairpin stem to stabilize hairpinsduring propagation in bacteria, for example. However, complementaritybetween the portion of the stem that binds to the target mRNA (antisensestrand) and the mRNA is typically required, and even a single base pairmismatch is this region may abolish silencing. 5′ and 3′ overhangs arenot required, since they do not appear to be critical for shRNAfunction, although they may be present (Paddison et al. (2002) Genes &Dev. 16(8):948-58).

MicroRNAs

Micro RNAs (miRNAs) are a highly conserved class of small RNA moleculesthat are transcribed from DNA in the genomes of plants and animals, butare not translated into protein. Processed miRNAs are single stranded˜17-25 nucleotide (nt) RNA molecules that become incorporated into theRNA-induced silencing complex (RISC) and have been identified as keyregulators of development, cell proliferation, apoptosis anddifferentiation. They are believed to play a role in regulation of geneexpression by binding to the 3′-untranslated region of specific mRNAs.RISC mediates down-regulation of gene expression through translationalinhibition, transcript cleavage, or both. RISC is also implicated intranscriptional silencing in the nucleus of a wide range of eukaryotes.

The number of miRNA sequences identified to date is large and growing,illustrative examples of which can be found, for example, in:Griffiths-Jones S, et al. NAR, 2006, 34, Database Issue, D140-D144;Griffiths-Jones S. NAR, 2004, 32, Database Issue, D109-D111; and also athttp://microrna.sanger.ac.uk/sequences/.

Antisense Oligonucleotides

In one embodiment, a nucleic acid is an antisense oligonucleotidedirected to a target polynucleotide. The term “antisenseoligonucleotide” or simply “antisense” is meant to includeoligonucleotides that are complementary to a targeted polynucleotidesequence. Antisense oligonucleotides are single strands of DNA or RNAthat are complementary to a chosen sequence. In the case of antisenseRNA, they prevent translation of complementary RNA strands by binding toit. Antisense DNA can be used to target a specific, complementary(coding or non-coding) RNA. If binding takes places this DNA/RNA hybridcan be degraded by the enzyme RNase H. In particular embodiment,antisense oligonucleotides contain from about 10 to about 50nucleotides, more preferably about 15 to about 30 nucleotides. The termalso encompasses antisense oligonucleotides that may not be exactlycomplementary to the desired target gene. Thus, the invention can beutilized in instances where non-target specific-activities are foundwith antisense, or where an antisense sequence containing one or moremismatches with the target sequence is the most preferred for aparticular use.

Antisense oligonucleotides have been demonstrated to be effective andtargeted inhibitors of protein synthesis, and, consequently, can be usedto specifically inhibit protein synthesis by a targeted gene. Theefficacy of antisense oligonucleotides for inhibiting protein synthesisis well established. For example, the synthesis of polygalactauronaseand the muscarine type 2 acetylcholine receptor are inhibited byantisense oligonucleotides directed to their respective mRNA sequences(U.S. Pat. No. 5,739,119 and U.S. Pat. No. 5,759,829). Further, examplesof antisense inhibition have been demonstrated with the nuclear proteincyclin, the multiple drug resistance gene (MDG1), ICAM-1, E-selectin,STK-1, striatal GABA_(A) receptor and human EGF (Jaskulski et al.,Science. 1988 Jun. 10; 240(4858):1544-6; Vasanthakumar and Ahmed, CancerCommun. 1989; 1(4):225-32; Penis et al., Brain Res Mol Brain Res. 1998Jun. 15; 57(2):310-20; U.S. Pat. No. 5,801,154; U.S. Pat. No. 5,789,573;U.S. Pat. No. 5,718,709 and U.S. Pat. No. 5,610,288). Furthermore,antisense constructs have also been described that inhibit and can beused to treat a variety of abnormal cellular proliferations, e.g. cancer(U.S. Pat. No. 5,747,470; U.S. Pat. No. 5,591,317 and U.S. Pat. No.5,783,683).

Methods of producing antisense oligonucleotides are known in the art andcan be readily adapted to produce an antisense oligonucleotide thattargets any polynucleotide sequence. Selection of antisenseoligonucleotide sequences specific for a given target sequence is basedupon analysis of the chosen target sequence and determination ofsecondary structure, T_(m), binding energy, and relative stability.Antisense oligonucleotides may be selected based upon their relativeinability to form dimers, hairpins, or other secondary structures thatwould reduce or prohibit specific binding to the target mRNA in a hostcell. Highly preferred target regions of the mRNA include those regionsat or near the AUG translation initiation codon and those sequences thatare substantially complementary to 5′ regions of the mRNA. Thesesecondary structure analyses and target site selection considerationscan be performed, for example, using v.4 of the OLIGO primer analysissoftware (Molecular Biology Insights) and/or the BLASTN 2.0.5 algorithmsoftware (Altschul et al., Nucleic Acids Res. 1997, 25(17):3389-402).

Ribozymes

According to another embodiment of the invention, nucleic acid-lipidparticles are associated with ribozymes. Ribozymes are RNA-proteincomplexes having specific catalytic domains that possess endonucleaseactivity (Kim and Cech, Proc Natl Acad Sci USA. 1987 December;84(24):8788-92; Forster and Symons, Cell. 1987 Apr. 24; 49(2):211-20).For example, a large number of ribozymes accelerate phosphoestertransfer reactions with a high degree of specificity, often cleavingonly one of several phosphoesters in an oligonucleotide substrate (Cechet al., Cell. 1981 December; 27(3 Pt 2):487-96; Michel and Westhof, JMol. Biol. 1990 Dec. 5; 216(3):585-610; Reinhold-Hurek and Shub, Nature.1992 May 14; 357(6374):173-6). This specificity has been attributed tothe requirement that the substrate bind via specific base-pairinginteractions to the internal guide sequence (“IGS”) of the ribozymeprior to chemical reaction.

At least six basic varieties of naturally-occurring enzymatic RNAs areknown presently. Each can catalyze the hydrolysis of RNA phosphodiesterbonds in trans (and thus can cleave other RNA molecules) underphysiological conditions. In general, enzymatic nucleic acids act byfirst binding to a target RNA. Such binding occurs through the targetbinding portion of an enzymatic nucleic acid which is held in closeproximity to an enzymatic portion of the molecule that acts to cleavethe target RNA. Thus, the enzymatic nucleic acid first recognizes andthen binds a target RNA through complementary base-pairing, and oncebound to the correct site, acts enzymatically to cut the target RNA.Strategic cleavage of such a target RNA will destroy its ability todirect synthesis of an encoded protein. After an enzymatic nucleic acidhas bound and cleaved its RNA target, it is released from that RNA tosearch for another target and can repeatedly bind and cleave newtargets.

The enzymatic nucleic acid molecule may be formed in a hammerhead,hairpin, a hepatitis δ virus, group I intron or RNaseP RNA (inassociation with an RNA guide sequence) or Neurospora VS RNA motif, forexample. Specific examples of hammerhead motifs are described by Rossiet al, Nucleic Acids Res. 1992 Sep. 11; 20(17):4559-65. Examples ofhairpin motifs are described by Hampel et al. (Eur. Pat. Appl. Publ. No.EP 0360257), Hampel and Tritz, Biochemistry 1989 Jun. 13;28(12):4929-33; Hampel et al., Nucleic Acids Res. 1990 Jan. 25;18(2):299-304 and U.S. Pat. No. 5,631,359. An example of the hepatitis δvirus motif is described by Perrotta and Been, Biochemistry. 1992 Dec.1; 31(47):11843-52; an example of the RNaseP motif is described byGuerrier-Takada et al., Cell. 1983 December; 35(3 Pt 2):849-57;Neurospora VS RNA ribozyme motif is described by Collins (Saville andCollins, Cell. 1990 May 18; 61(4):685-96; Saville and Collins, Proc NatlAcad Sci USA. 1991 Oct. 1; 88(19):8826-30; Collins and Olive,Biochemistry. 1993 Mar. 23; 32(11):2795-9); and an example of the GroupI intron is described in U.S. Pat. No. 4,987,071. Importantcharacteristics of enzymatic nucleic acid molecules used according tothe invention are that they have a specific substrate binding site whichis complementary to one or more of the target gene DNA or RNA regions,and that they have nucleotide sequences within or surrounding thatsubstrate binding site which impart an RNA cleaving activity to themolecule. Thus the ribozyme constructs need not be limited to specificmotifs mentioned herein.

Methods of producing a ribozyme targeted to any polynucleotide sequenceare known in the art. Ribozymes may be designed as described in Int.Pat. Appl. Publ. No. WO 93/23569 and Int. Pat. Appl. Publ. No. WO94/02595, each specifically incorporated herein by reference, andsynthesized to be tested in vitro and in vivo, as described therein.

Ribozyme activity can be optimized by altering the length of theribozyme binding arms or chemically synthesizing ribozymes withmodifications that prevent their degradation by serum ribonucleases (seee.g., Int. Pat. Appl. Publ. No. WO 92/07065; Int. Pat. Appl. Publ. No.WO 93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; Eur. Pat. Appl.Publ. No. 92110298.4; U.S. Pat. No. 5,334,711; and Int. Pat. Appl. Publ.No. WO 94/13688, which describe various chemical modifications that canbe made to the sugar moieties of enzymatic RNA molecules), modificationswhich enhance their efficacy in cells, and removal of stem II bases toshorten RNA synthesis times and reduce chemical requirements.

Immunostimulatory Oligonucleotides

Nucleic acids associated with lipid particles of the present inventionmay be immunostimulatory, including immunostimulatory oligonucleotides(ISS; single- or double-stranded) capable of inducing an immune responsewhen administered to a subject, which may be a mammal or other patient.ISS include, e.g., certain palindromes leading to hairpin secondarystructures (see Yamamoto S., et al. (1992) J. Immunol. 148: 4072-4076),or CpG motifs, as well as other known ISS features (such as multi-Gdomains, see WO 96/11266).

The immune response may be an innate or an adaptive immune response. Theimmune system is divided into a more innate immune system, and acquiredadaptive immune system of vertebrates, the latter of which is furtherdivided into humoral cellular components. In particular embodiments, theimmune response may be mucosal.

In particular embodiments, an immunostimulatory nucleic acid is onlyimmunostimulatory when administered in combination with a lipidparticle, and is not immunostimulatory when administered in its “freeform.” According to the present invention, such an oligonucleotide isconsidered to be immunostimulatory.

Immunostimulatory nucleic acids are considered to be non-sequencespecific when it is not required that they specifically bind to andreduce the expression of a target polynucleotide in order to provoke animmune response. Thus, certain immunostimulatory nucleic acids maycomprise a sequence corresponding to a region of a naturally occurringgene or mRNA, but they may still be considered non-sequence specificimmunostimulatory nucleic acids.

In one embodiment, the immunostimulatory nucleic acid or oligonucleotidecomprises at least one CpG dinucleotide. The oligonucleotide or CpGdinucleotide may be unmethylated or methylated. In another embodiment,the immunostimulatory nucleic acid comprises at least one CpGdinucleotide having a methylated cytosine. In one embodiment, thenucleic acid comprises a single CpG dinucleotide, wherein the cytosinein said CpG dinucleotide is methylated. In a specific embodiment, thenucleic acid comprises the sequence 5′ TAACGTTGAGGGGCAT 3′ (SEQ IDNO:2). In an alternative embodiment, the nucleic acid comprises at leasttwo CpG dinucleotides, wherein at least one cytosine in the CpGdinucleotides is methylated. In a further embodiment, each cytosine inthe CpG dinucleotides present in the sequence is methylated. In anotherembodiment, the nucleic acid comprises a plurality of CpG dinucleotides,wherein at least one of said CpG dinucleotides comprises a methylatedcytosine. Exemplary immunostimulatory oligonucleotides are shown inTable 1.

In one specific embodiment, the nucleic acid comprises the sequence 5′TTCCATGACGTTCCTGACGT 3′ (SEQ ID NO:1). In another specific embodiment,the nucleic acid sequence comprises the sequence 5′ TCCATGACGTTCCTGACGT3′ (SEQ ID NO:31), wherein the two cytosines indicated in bold aremethylated. In particular embodiments, the ODN is selected from a groupof ODNs consisting of ODN #1, ODN #2, ODN #3, ODN #4, ODN #5, ODN #6,ODN #7, ODN #8, and ODN #9, as shown below.

TABLE 1  Exemplary Immunostimulatory Oligonucleotides (ODNs) ODN NAMEODN SEQ ID NO ODN SEQUENCE (5′-3′). ODN 1 (INX-6295) SEQ ID NO: 25′-TAACGTTGAGGGGCAT-3 human c-myc * ODN 1m (INX-6303) SEQ ID NO: 45′-TAAZGTTGAGGGGCAT-3 ODN 2 (INX-1826) SEQ ID NO: 15′-TCCATGACGTTCCTGACGTT-3 * ODN 2m (INX-1826m) SEQ ID NO: 315′-TCCATGAZGTTCCTGAZGTT-3 ODN 3 (INX-6300) SEQ ID NO: 35′-TAAGCATACGGGGTGT-3 ODN 5 (INX-5001) SEQ ID NO: 5 5′-AACGTT-3ODN 6 (INX-3002) SEQ ID NO: 6 5′-GATGCTGTGTCGGGGTCTCCGGGC-3′ODN 7 (INX-2006) SEQ ID NO: 7 5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′ODN 7m (INX-2006m) SEQ ID NO: 32 5′-TZGTZGTTTTGTZGTTTTGTZGTT-3′ODN 8 (INX-1982) SEQ ID NO: 8 5′-TCCAGGACTTCTCTCAGGTT-3′ODN 9 (INX-G3139) SEQ ID NO: 9 5′-TCTCCCAGCGTGCGCCAT-3′ ODN 10 (PS-3082)SEQ ID NO: 10 5′-TGCATCCCCCAGGCCACCAT-3 murine IntracellularAdhesion Molecule-1 ODN 11 (PS-2302) SEQ ID NO: 115′-GCCCAAGCTGGCATCCGTCA-3′ human Intracellular Adhesion Molecule-1ODN 12 (PS-8997) SEQ ID NO: 12 5′-GCCCAAGCTGGCATCCGTCA-3′human Intracellular Adhesion Molecule-1 ODN 13 (US3) SEQ ID NO: 135′-GGT GCTCACTGC GGC-3′ human erb-B-2 ODN 14 (LR-3280) SEQ ID NO: 145′-AACC GTT GAG GGG CAT-3′ human c-myc ODN 15 (LR-3001) SEQ ID NO: 155′-TAT GCT GTG CCG GGG TCT TCG human c-myc GGC-3′ ODN 16 (Inx-6298)SEQ ID NO: 16 5′-GTGCCG GGGTCTTCGGGC-3′ ODN 17 (hIGF-1R) SEQ ID NO: 175′-GGACCCTCCTCCGGAGCC-3′ human Insulin  Growth Factor  1-ReceptorODN 18 (LR-52) SEQ ID NO: 18 5′-TCC TCC GGA GCC AGA CTT-3′human Insulin  Growth Factor  1-Receptor ODN 19 (hEGFR) SEQ ID NO: 195′-AAC GTT GAG GGG CAT-3′ human Epidermal Growth Factor- ReceptorODN 20 (EGFR) SEQ ID NO: 20 5′-CCGTGGTCA TGCTCC-3′ Epidermal GrowthFactor-Receptor ODN 21 (hVEGF) SEQ ID NO: 215′-CAG CCTGGCTCACCG CCTTGG-3′ human Vascular Endothelial Growth FactorODN 22 (PS-4189) SEQ ID NO: 22 5′-CAG CCA TGG TTC CCC CCA AC-3′ murinePhosphokinase  C-alpha ODN 23 (PS-3521) SEQ ID NO: 235′-GTT CTC GCT GGT GAG TTT CA-3′ ODN 24 (hBcl-2) SEQ ID NO: 245′-TCT CCCAGCGTGCGCCAT-3′ human Bcl-2 ODN 25 (hC-Raf-1)  SEQ ID NO: 255′-GTG CTC CAT TGA TGC-3′ human C-Raf-s ODN #26 (hVEGF-R1) SEQ ID NO: 265′-GAGUUCUGAUGAGGCCGAAAGGCCGAAAGUCUG-3′ human VascularEndothelial Growth Factor Receptor-1 ODN #27 SEQ ID NO: 27 5′-RRCGYY-3′ODN #28 (INX-3280).  SEQ ID NO: 28 5′-AACGTTGAGGGGCAT-3′ODN #29 (INX-6302) SEQ ID NO: 29 5′-CAACGTTATGGGGAGA-3′ODN #30 (INX-6298) SEQ ID NO: 30 5′-TAACGTTGAGGGGCAT-3′ human c-myc “Z”represents a methylated cytosine residue. Note: ODN 14 is a 15-meroligonucleotide and ODN 1 is the same oligonucleotide having a thymidineadded onto the 5′ end making ODN 1 into a 16-mer. No difference inbiological activity between ODN 14 and ODN 1 has been detected and bothexhibit similar immunostimulatory activity (Mui et al., J Pharmacol.Exp. Ther. 298: 1185-1192 (2001)).

Additional specific nucleic acid sequences of oligonucleotides (ODNs)suitable for use in the compositions and methods of the invention aredescribed in U.S. Patent Appln. 60/379,343, U.S. patent application Ser.No. 09/649,527, Int. Publ. WO 02/069369, Int. Publ. No. WO 01/15726,U.S. Pat. No. 6,406,705, and Raney et al., Journal of Pharmacology andExperimental Therapeutics, 298:1185-1192 (2001). In certain embodiments,ODNs used in the compositions and methods of the present invention havea phosphodiester (“PO”) backbone or a phosphorothioate (“PS”) backbone,and/or at least one methylated cytosine residue in a CpG motif.

Nucleic Acid Modifications

In the 1990's, DNA-based antisense oligodeoxynucleotides (ODN) andribozymes (RNA) represented an exciting new paradigm for drug design anddevelopment, but their application in vivo was prevented by endo- andexo-nuclease activity as well as a lack of successful intracellulardelivery. The degradation issue was effectively overcome followingextensive research into chemical modifications that prevented theoligonucleotide (oligo) drugs from being recognized by nuclease enzymesbut did not inhibit their mechanism of action. This research was sosuccessful that antisense ODN drugs in development today remain intactin vivo for days compared to minutes for unmodified molecules (Kurreck,J. 2003, Eur J Biochem 270:1628-44). However, intracellular delivery andmechanism of action issues have so far limited antisense ODN andribozymes from becoming clinical products.

RNA duplexes are inherently more stable to nucleases than singlestranded DNA or RNA, and unlike antisense ODN, unmodified siRNA showgood activity once they access the cytoplasm. Even so, the chemicalmodifications developed to stabilize antisense ODN and ribozymes havealso been systematically applied to siRNA to determine how much chemicalmodification can be tolerated and if pharmacokinetic and pharmacodynamicactivity can be enhanced. RNA interference by siRNA duplexes requires anantisense and sense strand, which have different functions. Both arenecessary to enable the siRNA to enter RISC, but once loaded the twostrands separate and the sense strand is degraded whereas the antisensestrand remains to guide RISC to the target mRNA. Entry into RISC is aprocess that is structurally less stringent than the recognition andcleavage of the target mRNA. Consequently, many different chemicalmodifications of the sense strand are possible, but only limited changesare tolerated by the antisense strand.

As is known in the art, a nucleoside is a base-sugar combination.Nucleotides are nucleosides that further include a phosphate groupcovalently linked to the sugar portion of the nucleoside. For thosenucleosides that include a pentofuranosyl sugar, the phosphate group canbe linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. Informing oligonucleotides, the phosphate groups covalently link adjacentnucleosides to one another to form a linear polymeric compound. In turnthe respective ends of this linear polymeric structure can be furtherjoined to form a circular structure. Within the oligonucleotidestructure, the phosphate groups are commonly referred to as forming theinternucleoside backbone of the oligonucleotide. The normal linkage orbackbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

The nucleic acid that is used in a lipid-nucleic acid particle accordingto this invention includes any form of nucleic acid that is known. Thus,the nucleic acid may be a modified nucleic acid of the type usedpreviously to enhance nuclease resistance and serum stability.Surprisingly, however, acceptable therapeutic products can also beprepared using the method of the invention to formulate lipid-nucleicacid particles from nucleic acids that have no modification to thephosphodiester linkages of natural nucleic acid polymers, and the use ofunmodified phosphodiester nucleic acids (i.e., nucleic acids in whichall of the linkages are phosphodiester linkages) is a preferredembodiment of the invention.

a. Backbone Modifications

Antisense, siRNA, and other oligonucleotides useful in this inventioninclude, but are not limited to, oligonucleotides containing modifiedbackbones or non-natural internucleoside linkages. Oligonucleotideshaving modified backbones include those that retain a phosphorus atom inthe backbone and those that do not have a phosphorus atom in thebackbone. Modified oligonucleotides that do not have a phosphorus atomin their internucleoside backbone can also be considered to beoligonucleosides. Modified oligonucleotide backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotri-esters,methyl and other alkyl phosphonates including 3′-alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates including3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, phosphoroselenate, methylphosphonate, orO-alkyl phosphotriester linkages, and boranophosphates having normal3′-5′ linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. Particular non-limiting examples ofparticular modifications that may be present in a nucleic acid accordingto the present invention are shown in Table 2.

Various salts, mixed salts and free acid forms are also included.Representative United States patents that teach the preparation of theabove linkages include, but are not limited to, U.S. Pat. Nos.3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897;5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676;5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126;5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and5,625,050.

In certain embodiments, modified oligonucleotide backbones that do notinclude a phosphorus atom therein have backbones that are formed byshort chain alkyl or cycloalkyl internucleoside linkages, mixedheteroatom and alkyl or cycloalkyl internucleoside linkages, or one ormore short chain heteroatomic or heterocyclic internucleoside linkages.These include, e.g., those having morpholino linkages (formed in partfrom the sugar portion of a nucleoside); siloxane backbones; sulfide,sulfoxide and sulfone backbones; formacetyl and thioformacetylbackbones; methylene formacetyl and thioformacetyl backbones; alkenecontaining backbones; sulfamate backbones; methyleneimino andmethylenehydrazino backbones; sulfonate and sulfonamide backbones; amidebackbones; and others having mixed N, O, S and CH₂ component parts.Representative United States patents that describe the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439.

The phosphorothioate backbone modification (Table 2, #1), where anon-bridging oxygen in the phosphodiester bond is replaced by sulfur, isone of the earliest and most common means deployed to stabilize nucleicacid drugs against nuclease degradation. In general, it appears that PSmodifications can be made extensively to both siRNA strands without muchimpact on activity (Kurreck, J., Eur. J. Biochem. 270:1628-44, 2003).However, PS oligos are known to avidly associate non-specifically withproteins resulting in toxicity, especially upon i.v. administration.Therefore, the PS modification is usually restricted to one or two basesat the 3′ and 5′ ends. The boranophosphate linker (Table 2, #2) is arecent modification that is apparently more stable than PS, enhancessiRNA activity and has low toxicity (Hall et al., Nucleic Acids Res.32:5991-6000, 2004).

TABLE 2 Chemical Modifications Applied to siRNA and Other Nucleic AcidsAbbrevi- Modification # ation Name Site Structure 1 PS PhosphorothioateBackbone

2 PB Boranophosphate Backbone

3 N3-MU N3-methyl-uridine Base

4 5′-BU 5′-bromo-uracil Base

5 5′-IU 5′-iodo-uracil Base

6 2,6-DP 2,6- diaminopurine Base

7 2′-F 2′-Fluoro Sugar

8 2′-OME 2″-O-methyl Sugar

9 2′-O- MOE 2′-O-(2- methoxylethyl) Sugar

10 2′-DNP 2′-O-(2,4- dinitrophenyl) Sugar

11 LNA Locked Nucleic Acid (methylene bridge connecting the 2′-oxygenwith the 4′-carbon of the ribose ring) Sugar

12 2′-Amino 2′-Amino Sugar

13 2′- Deoxy 2′-Deoxy Sugar

14 4′-thio 4′-thio- ribonucleotide Sugar

Other useful nucleic acids derivatives include those nucleic acidsmolecules in which the bridging oxygen atoms (those forming thephosphoester linkages) have been replaced with —S—, —NH—, —CH2- and thelike. In certain embodiments, the alterations to the antisense, siRNA,or other nucleic acids used will not completely affect the negativecharges associated with the nucleic acids. Thus, the present inventioncontemplates the use of antisense, siRNA, and other nucleic acids inwhich a portion of the linkages are replaced with, for example, theneutral methyl phosphonate or phosphoramidate linkages. When neutrallinkages are used, in certain embodiments, less than 80% of the nucleicacid linkages are so substituted, or less than 50% of the linkages areso substituted.

b. Base Modifications

Base modifications are less common than those to the backbone and sugar.The modifications shown in 0.3-6 all appear to stabilize siRNA againstnucleases and have little effect on activity (Zhang, H. Y., et al., CurrTop Med Chem 6:893-900 (2006)).

Accordingly, oligonucleotides may also include nucleobase (oftenreferred to in the art simply as “base”) modifications or substitutions.As used herein, “unmodified” or “natural” nucleobases include the purinebases adenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C orm5c), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine.

Certain nucleobases are particularly useful for increasing the bindingaffinity of the oligomeric compounds of the invention, including5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds., Antisense Research andApplications 1993, CRC Press, Boca Raton, pages 276-278). These may becombined, in particular embodiments, with 2′-O-methoxyethyl sugarmodifications. United States patents that teach the preparation ofcertain of these modified nucleobases as well as other modifiednucleobases include, but are not limited to, the above noted U.S. Pat.No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; and 5,681,941.

c. Sugar Modifications

Most modifications on the sugar group occur at the 2′-OH of the RNAsugar ring, which provides a convenient chemically reactive site(Manoharan, M., Curr Opin Chem Biol 8:570-9 (2004); Zhang, H. Y., etal., Curr Top Med Chem 6:893-900 (2006)). The 2′-F and 2′-OME are commonand both increase stability, the 2′-OME modification does not reduceactivity as long as it is restricted to less than 4 nucleotides perstrand (Holen, T., et al., Nucleic Acids Res 31:2401-7 (2003)). The2′-O-MOE is most effective in siRNA when modified bases are restrictedto the middle region of the molecule (Prakash, T. P., et al., J Med Chem48:4247-53 (2005)). Other modifications found to stabilize siRNA withoutloss of activity are shown in Table 2, 10-14.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. For example, the invention includes oligonucleotides thatcomprise one of the following at the 2′ position: OH; F; O-, S-, orN-alkyl, O-alkyl-O-alkyl, O-, S-, or N-alkenyl, or O-, S- or N-alkynyl,wherein the alkyl, alkenyl and alkynyl may be substituted orunsubstituted C₁ to C₁₀ alkyl or C2 to C10 alkenyl and alkynyl.Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃,O(CH₂)₂ON(CH₃)₂, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃,SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide, andother substituents having similar properties. One modification includes2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or2′-MOE) (Martin et al., Helv. Chim. Acta 1995, 78, 486-504), i.e., analkoxyalkoxy group. Other modifications include2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as2′-DMAOE, and 2′-dimethylaminoethoxyethoxy(2′-DMAEOE).

Additional modifications include 2′-methoxy(2′-O—CH₃),2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative United States patents that teach the preparationof such modified sugars structures include, but are not limited to, U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920.

In other oligonucleotide mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups, although the base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262. Further teaching of PNAcompounds can be found in Nielsen et al., Science 254, 1497-1500 (1991).

Particular embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂—(referred to as a methylene (methylimino) or MMI backbone)—CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂—(wherein the native phosphodiester backbone is represented as—O—P—O—CH₂—) of the above referenced U.S. Pat. No. 5,489,677, and theamide backbones of the above referenced U.S. Pat. No. 5,602,240. Alsopreferred are oligonucleotides having morpholino backbone structures ofthe above-referenced U.S. Pat. No. 5,034,506.

d. Chimeric Oligonucleotides

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. Certain preferredoligonucleotides of this invention are chimeric oligonucleotides.“Chimeric oligonucleotides” or “chimeras,” in the context of thisinvention, are oligonucleotides that contain two or more chemicallydistinct regions, each made up of at least one nucleotide. Theseoligonucleotides typically contain at least one region of modifiednucleotides that confers one or more beneficial properties (such as,e.g., increased nuclease resistance, increased uptake into cells,increased binding affinity for the RNA target) and a region that is asubstrate for RNase H cleavage.

In one embodiment, a chimeric oligonucleotide comprises at least oneregion modified to increase target binding affinity. Affinity of anoligonucleotide for its target is routinely determined by measuring theTm of an oligonucleotide/target pair, which is the temperature at whichthe oligonucleotide and target dissociate; dissociation is detectedspectrophotometrically. The higher the Tm, the greater the affinity ofthe oligonucleotide for the target. In one embodiment, the region of theoligonucleotide which is modified to increase target mRNA bindingaffinity comprises at least one nucleotide modified at the 2′ positionof the sugar, most preferably a 2′-O-alkyl, 2′-O-alkyl-O-alkyl or2′-fluoro-modified nucleotide. Such modifications are routinelyincorporated into oligonucleotides and these oligonucleotides have beenshown to have a higher Tm (i.e., higher target binding affinity) than2′-deoxyoligonucleotides against a given target. The effect of suchincreased affinity is to greatly enhance oligonucleotide inhibition oftarget gene expression.

In another embodiment, a chimeric oligonucleotide comprises a regionthat acts as a substrate for RNAse H. Of course, it is understood thatoligonucleotides may include any combination of the variousmodifications described herein

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. Such conjugates and methods of preparingthe same are known in the art.

Those skilled in the art will realize that for in vivo utility, such astherapeutic efficacy, a reasonable rule of thumb is that if a thiolatedversion of the sequence works in the free form, that encapsulatedparticles of the same sequence, of any chemistry, will also beefficacious. Encapsulated particles may also have a broader range of invivo utilities, showing efficacy in conditions and models not known tobe otherwise responsive to antisense therapy. Those skilled in the artknow that applying this invention they may find old models which nowrespond to antisense therapy. Further, they may revisit discardedantisense sequences or chemistries and find efficacy by employing theinvention.

The oligonucleotides used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including Applied Biosystems. Any other means for such synthesismay also be employed; the actual synthesis of the oligonucleotides iswell within the talents of the routineer. It is also well known to usesimilar techniques to prepare other oligonucleotides such as thephosphorothioates and alkylated derivatives.

Characteristic of Nucleic Acid-Lipid Particles

In certain embodiments, the present invention relates to methods andcompositions for producing lipid-encapsulated nucleic acid particles inwhich nucleic acids are encapsulated within a lipid layer. Such nucleicacid-lipid particles, incorporating siRNA oligonucleotides, arecharacterized using a variety of biophysical parameters including: (1)drug to lipid ratio; (2) encapsulation efficiency; and (3) particlesize. High drug to lipid rations, high encapsulation efficiency, goodnuclease resistance and serum stability and controllable particle size,generally less than 200 nm in diameter are desirable. In addition, thenature of the nucleic acid polymer is of significance, since themodification of nucleic acids in an effort to impart nuclease resistanceadds to the cost of therapeutics while in many cases providing onlylimited resistance. Unless stated otherwise, these criteria arecalculated in this specification as follows:

Nucleic acid to lipid ratio is the amount of nucleic acid in a definedvolume of preparation divided by the amount of lipid in the same volume.This may be on a mole per mole basis or on a weight per weight basis, oron a weight per mole basis. For final, administration-readyformulations, the nucleic acid:lipid ratio is calculated after dialysis,chromatography and/or enzyme (e.g., nuclease) digestion has beenemployed to remove as much of the external nucleic acid as possible;

Encapsulation efficiency refers to the drug to lipid ratio of thestarting mixture divided by the drug to lipid ratio of the final,administration competent formulation. This is a measure of relativeefficiency. For a measure of absolute efficiency, the total amount ofnucleic acid added to the starting mixture that ends up in theadministration competent formulation, can also be calculated. The amountof lipid lost during the formulation process may also be calculated.Efficiency is a measure of the wastage and expense of the formulation;and

Size indicates the size (diameter) of the particles formed. Sizedistribution may be determined using quasi-elastic light scattering(QELS) on a Nicomp Model 370 sub-micron particle sizer. Particles under200 nm are preferred for distribution to neo-vascularized (leaky)tissues, such as neoplasms and sites of inflammation.

Pharmaceutical Compositions

The lipid particles of present invention, particularly when associatedwith a therapeutic agent, may be formulated as a pharmaceuticalcomposition, e.g., which further comprises a pharmaceutically acceptablediluent, excipient, or carrier, such as physiological saline orphosphate buffer, selected in accordance with the route ofadministration and standard pharmaceutical practice.

In particular embodiments, pharmaceutical compositions comprising thelipid-nucleic acid particles of the invention are prepared according tostandard techniques and further comprise a pharmaceutically acceptablecarrier. Generally, normal saline will be employed as thepharmaceutically acceptable carrier. Other suitable carriers include,e.g., water, buffered water, 0.9% saline, 0.3% glycine, and the like,including glycoproteins for enhanced stability, such as albumin,lipoprotein, globulin, etc. In compositions comprising saline or othersalt containing carriers, the carrier is preferably added followinglipid particle formation. Thus, after the lipid-nucleic acidcompositions are formed, the compositions can be diluted intopharmaceutically acceptable carriers such as normal saline.

The resulting pharmaceutical preparations may be sterilized byconventional, well known sterilization techniques. The aqueous solutionscan then be packaged for use or filtered under aseptic conditions andlyophilized, the lyophilized preparation being combined with a sterileaqueous solution prior to administration. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, etc. Additionally, the lipidic suspension may includelipid-protective agents which protect lipids against free-radical andlipid-peroxidative damages on storage. Lipophilic free-radicalquenchers, such as α-tocopherol and water-soluble iron-specificchelators, such as ferrioxamine, are suitable.

The concentration of lipid particle or lipid-nucleic acid particle inthe pharmaceutical formulations can vary widely, i.e., from less thanabout 0.01%, usually at or at least about 0.05-5% to as much as 10 to30% by weight and will be selected primarily by fluid volumes,viscosities, etc., in accordance with the particular mode ofadministration selected. For example, the concentration may be increasedto lower the fluid load associated with treatment. This may beparticularly desirable in patients having atherosclerosis-associatedcongestive heart failure or severe hypertension. Alternatively,complexes composed of irritating lipids may be diluted to lowconcentrations to lessen inflammation at the site of administration. Inone group of embodiments, the nucleic acid will have an attached labeland will be used for diagnosis (by indicating the presence ofcomplementary nucleic acid). In this instance, the amount of complexesadministered will depend upon the particular label used, the diseasestate being diagnosed and the judgement of the clinician but willgenerally be between about 0.01 and about 50 mg per kilogram of bodyweight, preferably between about 0.1 and about 5 mg/kg of body weight.

As noted above, the nucleic acid-lipid particles of the invention mayinclude polyethylene glycol (PEG)-modified phospholipids, PEG-ceramide,or ganglioside G_(M1)-modified lipids or other lipids effective toprevent or limit aggregation. Addition of such components does notmerely prevent complex aggregation. Rather, it may also provide a meansfor increasing circulation lifetime and increasing the delivery of thelipid-nucleic acid composition to the target tissues.

The present invention also provides lipid-therapeutic agent compositionsin kit form. The kit will typically be comprised of a container that iscompartmentalized for holding the various elements of the kit. The kitwill contain the particles or pharmaceutical compositions of the presentinvention, preferably in dehydrated or concentrated form, withinstructions for their rehydration or dilution and administration. Incertain embodiments, the particles comprise the active agent, while inother embodiments, they do not.

D. Methods of Manufacture

The methods and compositions of the invention make use of certaincationic lipids, the synthesis, preparation and characterization ofwhich is described below and in the accompanying Examples. In addition,the present invention provides methods of preparing lipid particles,including those associated with a therapeutic agent, e.g., a nucleicacid. Generally, any method of preparing nucleic acid-lipid particlesmay be used according to the present invention by using one or more ofthe lipids of the present invention in the resulting nucleic acid-lipidparticles. Examples of suitable methods are known in the art anddescribed, e.g., in U.S. Patent Application Publication No.2006/0134189.

In one embodiment, a mixture of lipids is combined with a bufferedaqueous solution of nucleic acid to produce an intermediate mixturecontaining nucleic acid encapsulated in lipid particles wherein theencapsulated nucleic acids are present in a nucleic acid/lipid ratio ofabout 3 wt % to about 25 wt %, preferably 5 to 15 wt %. The intermediatemixture may optionally be sized to obtain lipid-encapsulated nucleicacid particles wherein the lipid portions are unilamellar vesicles,preferably having a diameter of 30 to 150 nm, more preferably about 40to 90 nm. The pH is then raised to neutralize at least a portion of thesurface charges on the lipid-nucleic acid particles, thus providing anat least partially surface-neutralized lipid-encapsulated nucleic acidcomposition.

As described above, several of these cationic lipids are amino lipidsthat are charged at a pH below the pK_(a) of the amino group andsubstantially neutral at a pH above the pK_(a). These cationic lipidsare termed titratable cationic lipids and can be used in theformulations of the invention using a two-step process. First, lipidvesicles can be formed at the lower pH with titratable cationic lipidsand other vesicle components in the presence of nucleic acids. In thismanner, the vesicles will encapsulate and entrap the nucleic acids.Second, the surface charge of the newly formed vesicles can beneutralized by increasing the pH of the medium to a level above thepK_(a) of the titratable cationic lipids present, i.e., to physiologicalpH or higher. Without intending to be bound by any particular theory, itis believed that the very high efficiency of nucleic acid encapsulationis a result of electrostatic interaction at low pH. At acidic pH (e.g.pH 4.0) the vesicle surface is charged and binds a portion of thenucleic acids through electrostatic interactions. When the externalacidic buffer is exchanged for a more neutral buffer (e.g. pH 7.5) thesurface of the lipid particle or liposome is neutralized, allowing anyexternal nucleic acid to be removed. More detailed information on theformulation process is provided in various publications (e.g., U.S. Pat.No. 6,287,591 and U.S. Pat. No. 6,858,225). Particularly advantageousaspects of this process include both the facile removal of any surfaceadsorbed nucleic acid and a resultant nucleic acid delivery vehiclewhich has a neutral surface. Liposomes or lipid particles having aneutral surface are expected to avoid rapid clearance from circulationand to avoid certain toxicities which are associated with cationicliposome preparations. It is further noted that the vesicles formed inthis manner provide formulations of uniform vesicle size with highcontent of nucleic acids. Additionally, the vesicles have a size rangeof from about 30 to about 150 nm, more preferably about 30 to about 90nm. Additional details concerning these uses of such titratable cationiclipids in the formulation of nucleic acid-lipid particles are providedin U.S. Pat. No. 6,287,591 and U.S. Pat. No. 6,858,225, incorporatedherein by reference.

In certain embodiments, the mixture of lipids includes at least twolipid components: a first amino lipid component of the present inventionthat is selected from among lipids which have a pKa such that the lipidis cationic at pH below the pKa and neutral at pH above the pKa, and asecond lipid component that is selected from among lipids that preventparticle aggregation during lipid-nucleic acid particle formation. Inparticular embodiments, the amino lipid is a novel cationic lipid of thepresent invention.

In certain embodiments of preparing the nucleic acid-lipid particles ofthe invention, the mixture of lipids is typically a solution of lipidsin an organic solvent. This mixture of lipids can then be dried to forma thin film or lyophilized to form a powder before being hydrated withan aqueous buffer to form liposomes. Alternatively, in a preferredmethod, the lipid mixture can be solubilized in a water misciblealcohol, such as ethanol, and this ethanolic solution added to anaqueous buffer resulting in spontaneous liposome formation. In mostembodiments, the alcohol is used in the form in which it is commerciallyavailable. For example, ethanol can be used as absolute ethanol (100%),or as 95% ethanol, the remainder being water. This method is describedin more detail in U.S. Pat. No. 5,976,567.

In one exemplary embodiment, the mixture of lipids is a mixture ofcationic amino lipids, neutral lipids (other than an amino lipid), asterol (e.g., cholesterol) and a PEG-modified lipid (e.g., a PEG-S-DMG,PEG-C-DOMG or PEG-DMA) in an alcohol solvent. In certain embodiments,the lipid mixture consists essentially of a cationic amino lipid, aneutral lipid, cholesterol and a PEG-modified lipid in alcohol, morepreferably ethanol. In certain embodiments, the first solution consistsof the above lipid mixture in molar ratios of about 20-70% amino lipid:5-45% neutral lipid:20-55% cholesterol:0.5-15% PEG-modified lipid. Inother embodiments, the first solution consists essentially ofDLin-K-C2-DMA, DSPC, Chol and PEG-S-DMG, PEG-C-DOMG or PEG-DMA, morepreferably in a molar ratio of about 20-60% DLin-K-C2-DMA: 5-25%DSPC:25-55% Chol:0.5-15% PEG-S-DMG, PEG-C-DOMG or PEG-DMA. In anothergroup of embodiments, the neutral lipid in these compositions isreplaced with POPC, DOPE or SM.

In certain embodiments in accordance with the invention, the lipidmixture is combined with a buffered aqueous solution that may containthe nucleic acids. The buffered aqueous solution of is typically asolution in which the buffer has a pH of less than the pK_(a) of theprotonatable lipid in the lipid mixture. Examples of suitable buffersinclude citrate, phosphate, acetate, and MES. A particularly preferredbuffer is citrate buffer. Preferred buffers will be in the range of1-1000 mM of the anion, depending on the chemistry of the nucleic acidbeing encapsulated, and optimization of buffer concentration may besignificant to achieving high loading levels (see, e.g., U.S. Pat. No.6,287,591 and U.S. Pat. No. 6,858,225). Alternatively, pure wateracidified to pH 5-6 with chloride, sulfate or the like may be useful. Inthis case, it may be suitable to add 5% glucose, or another non-ionicsolute which will balance the osmotic potential across the particlemembrane when the particles are dialyzed to remove ethanol, increase thepH, or mixed with a pharmaceutically acceptable carrier such as normalsaline. The amount of nucleic acid in buffer can vary, but willtypically be from about 0.01 mg/mL to about 200 mg/mL, more preferablyfrom about 0.5 mg/mL to about 50 mg/mL.

The mixture of lipids and the buffered aqueous solution of therapeuticnucleic acids is combined to provide an intermediate mixture. Theintermediate mixture is typically a mixture of lipid particles havingencapsulated nucleic acids. Additionally, the intermediate mixture mayalso contain some portion of nucleic acids which are attached to thesurface of the lipid particles (liposomes or lipid vesicles) due to theionic attraction of the negatively-charged nucleic acids andpositively-charged lipids on the lipid particle surface (the aminolipids or other lipid making up the protonatable first lipid componentare positively charged in a buffer having a pH of less than the pK_(a)of the protonatable group on the lipid). In one group of preferredembodiments, the mixture of lipids is an alcohol solution of lipids andthe volumes of each of the solutions is adjusted so that uponcombination, the resulting alcohol content is from about 20% by volumeto about 45% by volume. The method of combining the mixtures can includeany of a variety of processes, often depending upon the scale offormulation produced. For example, when the total volume is about 10-20mL or less, the solutions can be combined in a test tube and stirredtogether using a vortex mixer. Large-scale processes can be carried outin suitable production scale glassware.

Optionally, the lipid-encapsulated therapeutic agent (e.g., nucleicacid) complexes which are produced by combining the lipid mixture andthe buffered aqueous solution of therapeutic agents (nucleic acids) canbe sized to achieve a desired size range and relatively narrowdistribution of lipid particle sizes. Preferably, the compositionsprovided herein will be sized to a mean diameter of from about 70 toabout 200 nm, more preferably about 90 to about 130 nm. Severaltechniques are available for sizing liposomes to a desired size. Onesizing method is described in U.S. Pat. No. 4,737,323, incorporatedherein by reference. Sonicating a liposome suspension either by bath orprobe sonication produces a progressive size reduction down to smallunilamellar vesicles (SUVs) less than about 0.05 microns in size.Homogenization is another method which relies on shearing energy tofragment large liposomes into smaller ones. In a typical homogenizationprocedure, multilamellar vesicles are recirculated through a standardemulsion homogenizer until selected liposome sizes, typically betweenabout 0.1 and 0.5 microns, are observed. In both methods, the particlesize distribution can be monitored by conventional laser-beam particlesize determination. For certain methods herein, extrusion is used toobtain a uniform vesicle size.

Extrusion of liposome compositions through a small-pore polycarbonatemembrane or an asymmetric ceramic membrane results in a relativelywell-defined size distribution. Typically, the suspension is cycledthrough the membrane one or more times until the desired liposomecomplex size distribution is achieved. The liposomes may be extrudedthrough successively smaller-pore membranes, to achieve a gradualreduction in liposome size. In some instances, the lipid-nucleic acidcompositions which are formed can be used without any sizing.

In particular embodiments, methods of the present invention furthercomprise a step of neutralizing at least some of the surface charges onthe lipid portions of the lipid-nucleic acid compositions. By at leastpartially neutralizing the surface charges, unencapsulated nucleic acidis freed from the lipid particle surface and can be removed from thecomposition using conventional techniques. Preferably, unencapsulatedand surface adsorbed nucleic acids are removed from the resultingcompositions through exchange of buffer solutions. For example,replacement of a citrate buffer (pH about 4.0, used for forming thecompositions) with a HEPES-buffered saline (HBS pH about 7.5) solution,results in the neutralization of liposome surface and nucleic acidrelease from the surface. The released nucleic acid can then be removedvia chromatography using standard methods, and then switched into abuffer with a pH above the pKa of the lipid used.

Optionally the lipid vesicles (i.e., lipid particles) can be formed byhydration in an aqueous buffer and sized using any of the methodsdescribed above prior to addition of the nucleic acid, according to thepreformed vesicle (PFV) method. As described above, the aqueous buffershould be of a pH below the pKa of the amino lipid. A solution of thenucleic acids can then be added to these sized, preformed vesicles. Toallow encapsulation of nucleic acids into such “pre-formed” vesicles themixture should contain an alcohol, such as ethanol. In the case ofethanol, it should be present at a concentration of about 20% (w/w) toabout 45% (w/w). In addition, it may be necessary to warm the mixture ofpre-formed vesicles and nucleic acid in the aqueous buffer-ethanolmixture to a temperature of about 25° C. to about 50° C. depending onthe composition of the lipid vesicles and the nature of the nucleicacid. It will be apparent to one of ordinary skill in the art thatoptimization of the encapsulation process to achieve a desired level ofnucleic acid in the lipid vesicles will require manipulation of variablesuch as ethanol concentration and temperature. Examples of suitableconditions for nucleic acid encapsulation are provided in the Examples.Once the nucleic acids are encapsulated within the preformed vesicles,the external pH can be increased to at least partially neutralize thesurface charge. Unencapsulated and surface adsorbed nucleic acids canthen be removed as described above.

In other embodiments, nucleic acid lipid particles are prepared via acontinuous mixing method, e.g., a process that includes providing anaqueous solution comprising a nucleic acid such as an siRNA, in a firstreservoir, and providing an organic lipid solution in a secondreservoir, and mixing the aqueous solution with the organic lipidsolution such that the organic solution mixes with the aqueous solutionso as to substantially instantaneously produce a liposome encapsulatingthe nucleic acid. This process and the apparatus for carrying out thisprocess are described in detail in U.S. Patent Application PublicationNo. 2004/0142025.

In another embodiment, nucleic acid lipid particles are produced via adirect dilution process that includes forming a liposome solution andimmediately and directly introducing the liposome solution to acollection vessel containing a controlled amount of dilution buffer. Incertain embodiments, the collection vessel includes one or more elementsconfigured to stir the contents of the collection vessel to facilitatedilution. In other embodiment, a third reservoir containing dilutionbuffer is fluidly coupled to a second mixing region. In this embodiment,t liposome solution formed in the first mixing region is immediately anddirectly mixed with the dilution buffer in the second mixing region.Processes and apparati for carrying out these direct dilution methodsare described in further detail in U.S. Patent Application PublicationNo. 2007/0042031.

E. Method of Use

The lipid particles of the present invention may be used to deliver atherapeutic agent to a cell, in vitro or in vivo. In particularembodiments, the therapeutic agent is a nucleic acid, which is deliveredto a cell using a nucleic acid-lipid particles of the present invention.While the following description of various methods of using the lipidparticles and related pharmaceutical compositions of the presentinvention are exemplified by description related to nucleic acid-lipidparticles, it is understood that these methods and compositions may bereadily adapted for the delivery of any therapeutic agent for thetreatment of any disease or disorder that would benefit from suchtreatment.

In certain embodiments, the present invention provides methods forintroducing a nucleic acid into a cell. Preferred nucleic acids forintroduction into cells are siRNA, immune-stimulating oligonucleotides,plasmids, antisense oligonucleotides, and ribozymes. These methods maybe carried out by contacting the particles or compositions of thepresent invention with the cells for a period of time sufficient forintracellular delivery to occur.

The compositions of the present invention can be adsorbed to almost anycell type. Once adsorbed, the nucleic acid-lipid particles can either beendocytosed by a portion of the cells, exchange lipids with cellmembranes, or fuse with the cells. Transfer or incorporation of thenucleic acid portion of the complex can take place via any one of thesepathways. Without intending to be limited with respect to the scope ofthe invention, it is believed that in the case of particles taken upinto the cell by endocytosis the particles then interact with theendosomal membrane, resulting in destabilization of the endosomalmembrane, possibly by the formation of non-bilayer phases, resulting inintroduction of the encapsulated nucleic acid into the cell cytoplasm.Similarly in the case of direct fusion of the particles with the cellplasma membrane, when fusion takes place, the liposome membrane isintegrated into the cell membrane and the contents of the liposomecombine with the intracellular fluid. Contact between the cells and thelipid-nucleic acid compositions, when carried out in vitro, will takeplace in a biologically compatible medium. The concentration ofcompositions can vary widely depending on the particular application,but is generally between about 1 μmol and about 10 mmol. In certainembodiments, treatment of the cells with the lipid-nucleic acidcompositions will generally be carried out at physiological temperatures(about 37° C.) for periods of time from about 1 to 24 hours, preferablyfrom about 2 to 8 hours. For in vitro applications, the delivery ofnucleic acids can be to any cell grown in culture, whether of plant oranimal origin, vertebrate or invertebrate, and of any tissue or type. Inpreferred embodiments, the cells will be animal cells, more preferablymammalian cells, and most preferably human cells.

In one group of embodiments, a lipid-nucleic acid particle suspension isadded to 60-80% confluent plated cells having a cell density of fromabout 10³ to about 10⁵ cells/mL, more preferably about 2×10⁴ cells/mL.The concentration of the suspension added to the cells is preferably offrom about 0.01 to 20 μg/mL, more preferably about 1 μg/mL.

Typical applications include using well known procedures to provideintracellular delivery of siRNA to knock down or silence specificcellular targets. Alternatively applications include delivery of DNA ormRNA sequences that code for therapeutically useful polypeptides. Inthis manner, therapy is provided for genetic diseases by supplyingdeficient or absent gene products (i.e., for Duchenne's dystrophy, seeKunkel, et al., Brit. Med. Bull. 45(3):630-643 (1989), and for cysticfibrosis, see Goodfellow, Nature 341:102-103 (1989)). Other uses for thecompositions of the present invention include introduction of antisenseoligonucleotides in cells (see, Bennett, et al., Mol. Pharm.41:1023-1033 (1992)).

Alternatively, the compositions of the present invention can also beused for deliver of nucleic acids to cells in vivo, using methods whichare known to those of skill in the art. With respect to application ofthe invention for delivery of DNA or mRNA sequences, Zhu, et al.,Science 261:209-211 (1993), incorporated herein by reference, describesthe intravenous delivery of cytomegalovirus (CMV)-chloramphenicolacetyltransferase (CAT) expression plasmid using DOTMA-DOPE complexes.Hyde, et al., Nature 362:250-256 (1993), incorporated herein byreference, describes the delivery of the cystic fibrosis transmembraneconductance regulator (CFTR) gene to epithelia of the airway and toalveoli in the lung of mice, using liposomes. Brigham, et al., Am. J.Med. Sci. 298:278-281 (1989), incorporated herein by reference,describes the in vivo transfection of lungs of mice with a functioningprokaryotic gene encoding the intracellular enzyme, chloramphenicolacetyltransferase (CAT). Thus, the compositions of the invention can beused in the treatment of infectious diseases.

For in vivo administration, the pharmaceutical compositions arepreferably administered parenterally, i.e., intraarticularly,intravenously, intraperitoneally, subcutaneously, or intramuscularly. Inparticular embodiments, the pharmaceutical compositions are administeredintravenously or intraperitoneally by a bolus injection. For oneexample, see Stadler, et al., U.S. Pat. No. 5,286,634, which isincorporated herein by reference. Intracellular nucleic acid deliveryhas also been discussed in Straubringer, et al., METHODS IN ENZYMOLOGY,Academic Press, New York. 101:512-527 (1983); Mannino, et al.,Biotechniques 6:682-690 (1988); Nicolau, et al., Crit. Rev. Ther. DrugCarrier Syst. 6:239-271 (1989), and Behr, Acc. Chem. Res. 26:274-278(1993). Still other methods of administering lipid-based therapeuticsare described in, for example, Rahman et al., U.S. Pat. No. 3,993,754;Sears, U.S. Pat. No. 4,145,410; Papahadjopoulos et al., U.S. Pat. No.4,235,871; Schneider, U.S. Pat. No. 4,224,179; Lenk et al., U.S. Pat.No. 4,522,803; and Fountain et al., U.S. Pat. No. 4,588,578.

In other methods, the pharmaceutical preparations may be contacted withthe target tissue by direct application of the preparation to thetissue. The application may be made by topical, “open” or “closed”procedures. By “topical,” it is meant the direct application of thepharmaceutical preparation to a tissue exposed to the environment, suchas the skin, oropharynx, external auditory canal, and the like. “Open”procedures are those procedures which include incising the skin of apatient and directly visualizing the underlying tissue to which thepharmaceutical preparations are applied. This is generally accomplishedby a surgical procedure, such as a thoracotomy to access the lungs,abdominal laparotomy to access abdominal viscera, or other directsurgical approach to the target tissue. “Closed” procedures are invasiveprocedures in which the internal target tissues are not directlyvisualized, but accessed via inserting instruments through small woundsin the skin. For example, the preparations may be administered to theperitoneum by needle lavage. Likewise, the pharmaceutical preparationsmay be administered to the meninges or spinal cord by infusion during alumbar puncture followed by appropriate positioning of the patient ascommonly practiced for spinal anesthesia or metrazamide imaging of thespinal cord. Alternatively, the preparations may be administered throughendoscopic devices.

The lipid-nucleic acid compositions can also be administered in anaerosol inhaled into the lungs (see, Brigham, at al., Am. J. Sci.298(4):278-281 (1989)) or by direct injection at the site of disease(Culver, Human Gene Therapy, MaryAnn Liebert, Inc., Publishers, NewYork. pp. 70-71 (1994)).

The methods of the present invention may be practiced in a variety ofhosts. Preferred hosts include mammalian species, such as humans,non-human primates, dogs, cats, cattle, horses, sheep, and the like.

Dosages for the lipid-therapeutic agent particles of the presentinvention will depend on the ratio of therapeutic agent to lipid and theadministrating physician's opinion based on age, weight, and conditionof the patient.

In one embodiment, the present invention provides a method of modulatingthe expression of a target polynucleotide or polypeptide. These methodsgenerally comprise contacting a cell with a lipid particle of thepresent invention that is associated with a nucleic acid capable ofmodulating the expression of a target polynucleotide or polypeptide. Asused herein, the term “modulating” refers to altering the expression ofa target polynucleotide or polypeptide. In different embodiments,modulating can mean increasing or enhancing, or it can mean decreasingor reducing. Methods of measuring the level of expression of a targetpolynucleotide or polypeptide are known and available in the arts andinclude, e.g., methods employing reverse transcription-polymerase chainreaction (RT-PCR) and immunohistochemical techniques. In particularembodiments, the level of expression of a target polynucleotide orpolypeptide is increased or reduced by at least 10%, 20%, 30%, 40%, 50%,or greater than 50% as compared to an appropriate control value.

For example, if increased expression of a polypeptide desired, thenucleic acid may be an expression vector that includes a polynucleotidethat encodes the desired polypeptide. On the other hand, if reducedexpression of a polynucleotide or polypeptide is desired, then thenucleic acid may be, e.g., an antisense oligonucleotide, siRNA, ormicroRNA that comprises a polynucleotide sequence that specificallyhybridizes to a polynucleotide that encodes the target polypeptide,thereby disrupting expression of the target polynucleotide orpolypeptide. Alternatively, the nucleic acid may be a plasmid thatexpresses such an antisense oligonucleotide, siRNA, or microRNA.

In one particular embodiment, the present invention provides a method ofmodulating the expression of a polypeptide by a cell, comprisingproviding to a cell a lipid particle that consists of or consistsessentially of DLin-K-C2-DMA, DSPC, Chol and PEG-S-DMG, PEG-C-DOMG orPEG-DMA, e.g., in a molar ratio of about 20-60% DLin-K-C2-DMA: 5-25%DSPC:25-55% Chol:0.5-15% PEG-S-DMG, PEG-C-DOMG or PEG-DMA, wherein thelipid particle is associated with a nucleic acid capable of modulatingthe expression of the polypeptide. In particular embodiments, the molarlipid ratio is approximately 40/10/40/10 (mol %DLin-K-C2-DMA/DSPC/Chol/PEG-S-DMG). In another group of embodiments, theneutral lipid in these compositions is replaced with POPC, DOPE or SM.In other embodiments, the cationic lipid is replaced with DLin-K²-DMA orDLin-K6-DMA.

In particular embodiments, the therapeutic agent is selected from ansiRNA, a microRNA, an antisense oligonucleotide, and a plasmid capableof expressing an siRNA, a microRNA, or an antisense oligonucleotide, andwherein the siRNA, microRNA, or antisense RNA comprises a polynucleotidethat specifically binds to a polynucleotide that encodes thepolypeptide, or a complement thereof, such that the expression of thepolypeptide is reduced.

In other embodiments, the nucleic acid is a plasmid that encodes thepolypeptide or a functional variant or fragment thereof, such thatexpression of the polypeptide or the functional variant or fragmentthereof is increased.

In related embodiments, the present invention provides a method oftreating a disease or disorder characterized by overexpression of apolypeptide in a subject, comprising providing to the subject apharmaceutical composition of the present invention, wherein thetherapeutic agent is selected from an siRNA, a microRNA, an antisenseoligonucleotide, and a plasmid capable of expressing an siRNA, amicroRNA, or an antisense oligonucleotide, and wherein the siRNA,microRNA, or antisense RNA comprises a polynucleotide that specificallybinds to a polynucleotide that encodes the polypeptide, or a complementthereof.

In one embodiment, the pharmaceutical composition comprises a lipidparticle that consists of or consists essentially of DLin-K-C2-DMA,DSPC, Chol and PEG-S-DMG, PEG-C-DOMG or PEG-DMA, e.g., in a molar ratioof about 20-60% DLin-K-C2-DMA: 5-25% DSPC:25-55% Chol:0.5-15% PEG-S-DMG,PEG-C-DOMG or PEG-DMA, wherein the lipid particle is associated with thetherapeutic nucleic acid. In particular embodiments, the molar lipidratio is approximately 40/10/40/10 (mol %DLin-K-C2-DMA/DSPC/Chol/PEG-S-DMG). In another group of embodiments, theneutral lipid in these compositions is replaced with POPC, DOPE or SM.In other embodiments, the cationic lipid is replaced with DLin-K²-DMA orDLin-K6-DMA.

In another related embodiment, the present invention includes a methodof treating a disease or disorder characterized by underexpression of apolypeptide in a subject, comprising providing to the subject apharmaceutical composition of the present invention, wherein thetherapeutic agent is a plasmid that encodes the polypeptide or afunctional variant or fragment thereof.

In one embodiment, the pharmaceutical composition comprises a lipidparticle that consists of or consists essentially of DLin-K-C2-DMA,DSPC, Chol and PEG-S-DMG, PEG-C-DOMG or PEG-DMA, e.g., in a molar ratioof about 20-60% DLin-K-C2-DMA: 5-25% DSPC:25-55% Chol:0.5-15% PEG-S-DMGor PEG-DMA, wherein the lipid particle is associated with thetherapeutic nucleic acid. In particular embodiments, the molar lipidratio is approximately 40/10/40/10 (mol %DLin-K-C2-DMA/DSPC/Chol/PEG-S-DMG). In another group of embodiments, theneutral lipid in these compositions is replaced with POPC, DOPE or SM.In other embodiments, the cationic lipid is replaced with DLin-K²-DMA orDLin-K6-DMA.

The present invention further provides a method of inducing an immuneresponse in a subject, comprising providing to the subject thepharmaceutical composition of the present invention, wherein thetherapeutic agent is an immunostimulatory oligonucleotide. In certainembodiments, the immune response is a humoral or mucosal immuneresponse. In one embodiment, the pharmaceutical composition comprises alipid particle that consists of or consists essentially ofDLin-K-C2-DMA, DSPC, Chol and PEG-S-DMG, PEG-C-DOMG or PEG-DMA, e.g., ina molar ratio of about 20-60% DLin-K-C2-DMA: 5-25% DSPC:25-55%Chol:0.5-15% PEG-S-DMG, PEG-C-DOMG or PEG-DMA, wherein the lipidparticle is associated with the therapeutic nucleic acid. In particularembodiments, the molar lipid ratio is approximately 40/10/40/10 (mol %DLin-K-C2-DMA/DSPC/Chol/PEG-S-DMG, PEG-C-DOMG or PEG-DMA). In anothergroup of embodiments, the neutral lipid in these compositions isreplaced with POPC, DOPE or SM. In other embodiments, the cationic lipidis replaced with DLin-K²-DMA or DLin-K6-DMA.

In further embodiments, the pharmaceutical composition is provided tothe subject in combination with a vaccine or antigen. Thus, the presentinvention itself provides vaccines comprising a lipid particle of thepresent invention, which comprises an immunostimulatory oligonucleotide,and is also associated with an antigen to which an immune response isdesired. In particular embodiments, the antigen is a tumor antigen or isassociated with an infective agent, such as, e.g., a virus, bacteria, orparasite.

A variety of tumor antigens, infections agent antigens, and antigensassociated with other disease are well known in the art and examples ofthese are described in references cited herein. Examples of antigenssuitable for use in the present invention include, but are not limitedto, polypeptide antigens and DNA antigens. Specific examples of antigensare Hepatitis A, Hepatitis B, small pox, polio, anthrax, influenza,typhus, tetanus, measles, rotavirus, diphtheria, pertussis,tuberculosis, and rubella antigens. In a preferred embodiment, theantigen is a Hepatitis B recombinant antigen. In other aspects, theantigen is a Hepatitis A recombinant antigen. In another aspect, theantigen is a tumor antigen. Examples of such tumor-associated antigensare MUC-1, EBV antigen and antigens associated with Burkitt's lymphoma.In a further aspect, the antigen is a tyrosinase-related protein tumorantigen recombinant antigen. Those of skill in the art will know ofother antigens suitable for use in the present invention.

Tumor-associated antigens suitable for use in the subject inventioninclude both mutated and non-mutated molecules that may be indicative ofsingle tumor type, shared among several types of tumors, and/orexclusively expressed or overexpressed in tumor cells in comparison withnormal cells. In addition to proteins and glycoproteins, tumor-specificpatterns of expression of carbohydrates, gangliosides, glycolipids andmucins have also been documented. Exemplary tumor-associated antigensfor use in the subject cancer vaccines include protein products ofoncogenes, tumor suppressor genes and other genes with mutations orrearrangements unique to tumor cells, reactivated embryonic geneproducts, oncofetal antigens, tissue-specific (but not tumor-specific)differentiation antigens, growth factor receptors, cell surfacecarbohydrate residues, foreign viral proteins and a number of other selfproteins.

Specific embodiments of tumor-associated antigens include, e.g., mutatedantigens such as the protein products of the Ras p21 protooncogenes,tumor suppressor p53 and BCR-abl oncogenes, as well as CDK4, MUM1,Caspase 8, and Beta catenin; overexpressed antigens such as galectin 4,galectin 9, carbonic anhydrase, Aldolase A, PRAME, Her2/neu, ErbB-2 andKSA, oncofetal antigens such as alpha fetoprotein (AFP), human chorionicgonadotropin (hCG); self antigens such as carcinoembryonic antigen (CEA)and melanocyte differentiation antigens such as Mart 1/Melan A, gp100,gp75, Tyrosinase, TRP1 and TRP2; prostate associated antigens such asPSA, PAP, PSMA, PSM-P1 and PSM-P2; reactivated embryonic gene productssuch as MAGE 1, MAGE 3, MAGE 4, GAGE 1, GAGE 2, BAGE, RAGE, and othercancer testis antigens such as NY-ESO1, SSX2 and SCP1; mucins such asMuc-1 and Muc-2; gangliosides such as GM2, GD2 and GD3, neutralglycolipids and glycoproteins such as Lewis (y) and globo-H; andglycoproteins such as Tn, Thompson-Freidenreich antigen (TF) and sTn.Also included as tumor-associated antigens herein are whole cell andtumor cell lysates as well as immunogenic portions thereof, as well asimmunoglobulin idiotypes expressed on monoclonal proliferations of Blymphocytes for use against B cell lymphomas.

Pathogens include, but are not limited to, infectious agents, e.g.,viruses, that infect mammals, and more particularly humans. Examples ofinfectious virus include, but are not limited to: Retroviridae (e.g.,human immunodeficiency viruses, such as HIV-1 (also referred to asHTLV-III, LAV or HTLV-III/LAV, or HIV-III; and other isolates, such asHIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A virus;enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses);Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae(e.g., equine encephalitis viruses, rubella viruses); Flaviridae (e.g.,dengue viruses, encephalitis viruses, yellow fever viruses);Coronoviridae (e.g., coronaviruses); Rhabdoviradae (e.g., vesicularstomatitis viruses, rabies viruses); Coronaviridae (e.g.,coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses,rabies viruses); Filoviridae (e.g., ebola viruses); Paramyxoviridae(e.g., parainfluenza viruses, mumps virus, measles virus, respiratorysyncytial virus); Orthomyxoviridae (e.g., influenza viruses);Bungaviridae (e.g., Hantaan viruses, bunga viruses, phleboviruses andNairo viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae(e.g., reoviruses, orbiviurses and rotaviruses); Birnaviridae;Hepadnaviridae (Hepatitis B virus); Parvovirida (parvoviruses);Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (mostadenoviruses); Herpesviridae herpes simplex virus (HSV) 1 and 2,varicella zoster virus, cytomegalovirus (CMV), herpes virus; Poxyviridae(variola viruses, vaccinia viruses, pox viruses); and Iridoviridae(e.g., African swine fever virus); and unclassified viruses (e.g., theetiological agents of Spongiform encephalopathies, the agent of deltahepatitis (thought to be a defective satellite of hepatitis B virus),the agents of non-A, non-B hepatitis (class 1=internally transmitted;class 2=parenterally transmitted (i.e., Hepatitis C); Norwalk andrelated viruses, and astroviruses).

Also, gram negative and gram positive bacteria serve as antigens invertebrate animals. Such gram positive bacteria include, but are notlimited to Pasteurella species, Staphylococci species, and Streptococcusspecies. Gram negative bacteria include, but are not limited to,Escherichia coli, Pseudomonas species, and Salmonella species. Specificexamples of infectious bacteria include but are not limited to:Helicobacterpyloris, Borelia burgdorferi, Legionella pneumophilia,Mycobacteria sps (e.g., M. tuberculosis, M. avium, M. intracellulare, M.kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae,Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes(Group A Streptococcus), Streptococcus agalactiae (Group BStreptococcus), Streptococcus (viridans group), Streptococcus faecalis,Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcuspneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilusinfluenzae, Bacillus antracis, corynebacterium diphtheriae,corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridiumperfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiellapneumoniae, Pasturella multocida, Bacteroides sp., Fusobacteriumnucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponemapertenue, Leptospira, Rickettsia, and Actinomyces israelli.

Additional examples of pathogens include, but are not limited to,infectious fungi that infect mammals, and more particularly humans.Examples of infectious fungi include, but are not limited to:Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis,Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans.Examples of infectious parasites include Plasmodium such as Plasmodiumfalciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax.Other infectious organisms (i.e., protists) include Toxoplasma gondii.

EXAMPLES Example 1 Synthesis of2,2-Dilinoleyl-4-Dimethylaminomethyl-[1,3]-Dioxolane (DLin-K-DMA)

DLin-K-DMA was synthesized as shown in the following schematic anddescribed below.

Synthesis of Linoleyl Bromide (II)

A mixture of linoleyl methane sulfonate (6.2 g, 18 mmol) and magnesiumbromide etherate (17 g, 55 mmol) in anhydrous ether (300 mL) was stirredunder argon overnight (21 hours). The resulting suspension was pouredinto 300 mL of chilled water. Upon shaking, the organic phase wasseparated. The aqueous phase was extracted with ether (2×150 mL). Thecombined ether phase was washed with water (2×150 mL), brine (150 mL),and dried over anhydrous Na₂SO₄. The solvent was evaporated to afford6.5 g of colourless oil. The crude product was purified by columnchromatography on silica gel (230-400 mesh, 300 mL) and eluted withhexanes. This gave 6.2 g (approximately 100%) of linoleyl bromide (II).¹H NMR (400 MHz, CDCl3) δ: 5.27-5.45 (4H, m, 2×CH═CH), 3.42 (2H, t,CH2Br), 2.79 (2H, t, C═C—CH2-C═C), 2.06 (4H, q, 2× allylic CH2), 1.87(2H, quintet, CH2), 1.2-1.5 (16H, m), 0.90 (3H, t, CH3) ppm.

Synthesis of Dilinoleyl Methanol (III)

To a suspension of Mg turnings (0.45 g, 18.7 mmol) with one crystal ofiodine in 200 mL of anhydrous ether under nitrogen was added a solutionof linoleyl bromide (II) in 50 mL of anhydrous ether at roomtemperature. The resulting mixture was refluxed under nitrogenovernight. The mixture was cooled to room temperature. To the cloudymixture under nitrogen was added dropwise at room temperature a solutionof ethyl formate (0.65 g, 18.7 mmol) in 30 mL of anhydrous ether. Uponaddition, the mixture was stirred at room temperature overnight (20hours). The ether layer was washed with 10% H₂SO₄ aqueous solution (100mL), water (2×100 mL), brine (150 mL), and then dried over anhydrousNa₂SO₄. Evaporation of the solvent gave 5.0 g of pale oil. Columnchromatography on silica gel (230-400 mesh, 300 mL) with 0-7% ethergradient in hexanes as eluent afforded two products, dilinoleyl methanol(2.0 g, III) and dilinoleylmethyl formate (1.4 g, IV). ¹H NMR (400 MHz,CDCl3) for dilinoleylmethyl formate (IV) δ: 8.10 (1H, s, CHO), 5.27-5.45(8H, m, 4×CH═CH), 4.99 (1H, quintet, OCH), 2.78 (4H, t, 2×C═C—CH2-C═C),2.06 (8H, q, 4×allylic CH2), 1.5-1.6 (4H, m, 2×CH2), 1.2-1.5 (32H, m),0.90 (6H, t, 2×CH3) ppm.

Dilinoleylmethyl formate (IV, 1.4 g) and KOH (0.2 g) were stirred in 85%EtOH at room temperature under nitrogen overnight. Upon completion ofthe reaction, half of the solvent was evaporated. The resulting mixturewas poured into 150 mL of 5% HCL solution. The aqueous phase wasextracted with ether (3×100 ml). The combined ether extract was washedwith water (2×100 mL), brine (100 mL), and dried over anhydrous Na2SO4.Evaporation of the solvent gave 1.0 g of dilinoleyl methanol (III) ascolourless oil. Overall, 3.0 g (60%) of dilinoleyl methanol (III) wereafforded. 1H NMR (400 MHz, CDCl3) for dilinoleyl methanol (III) δ: ppm.

Synthesis of Dilinoleyl Ketone (V)

To a mixture of dilinoleyl methanol (2.0 g, 3.8 mmol) and anhydroussodium carbonate (0.2 g) in 100 mL of CH₂Cl₂ was added pydimiumchlorochromate (PCC, 2.0 g, 9.5 mmol). The resulting suspension wasstirred at room temperature for 60 min. Ether (300 mL) was then addedinto the mixture, and the resulting brown suspension was filteredthrough a pad of silica gel (300 mL). The silica gel pad was furtherwashed with ether (3×200 mL). The ether filtrate and washes werecombined. Evaporation of the solvent gave 3.0 g of an oily residual as acrude product. The crude product was purified by column chromatographyon silica gel (230-400 mesh, 250 mL) eluted with 0-3% ether in hexanes.This gave 1.8 g (90%) of dilinoleyl ketone (V). ¹H NMR (400 MHz, CDCl3)δ: 5.25-5.45 (8H, m, 4×CH═CH), 2.78 (4H, t, 2×C═C—CH2-C═C), 2.39 (4H, t,2×COCH2), 2.05 (8H, q, 4×allylic CH2), 1.45-1.7 (4H, m), 1.2-1.45 (32H,m), 0.90 (6H, t, 2×CH3) ppm.

Synthesis of 2,2-Dilinoleyl-4-bromomethyl-[1,3]-dioxolane (VI)

A mixture of dilinoleyl methanol (V, 1.3 g, 2.5 mmol),3-bromo-1,2-propanediol (1.5 g, 9.7 mmol) and p-toluene sulfonic acidhydrate (0.16 g, 0.84 mmol) in 200 mL of toluene was refluxed undernitrogen for 3 days with a Dean-Stark tube to remove water. Theresulting mixture was cooled to room temperature. The organic phase waswashed with water (2×50 mL), brine (50 mL), and dried over anhydrousNa₂SO₄. Evaporation of the solvent resulted in a yellowish oily residue.Column chromatography on silica gel (230-400 mesh, 100 mL) with 0-6%ether gradient in hexanes as eluent afforded 0.1 g of pure VI and 1.3 gof a mixture of VI and the starting material. 1H NMR (400 MHz, CDCl3) δ:5.27-5.45 (8H, m, 4×CH═CH), 4.28-4.38 (1H, m, OCH), 4.15 (1H, dd, OCH),3.80 (1H, dd, OCH), 3.47 (1H, dd, CHBr), 3.30 (1H, dd, CHBr), 2.78 (4H,t, 2×C═C—CH2-C═C), 2.06 (8H, q, 4×allylic CH2), 1.52-1.68 (4H, m,2×CH2), 1.22-1.45 (32H, m), 0.86-0.94 (6H, m, 2×CH3) ppm.

Synthesis of 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane(DLin-K-DMA)

Anhydrous dimethyl amine was bubbled into an anhydrous THF solution (100mL) containing 1.3 g of a mixture of2,2-dilinoleyl-4-bromomethyl-[1,3]-dioxolane (VI) and dilinoleyl ketone(V) at 0° C. for 10 min. The reaction flask was then sealed and themixture stirred at room temperature for 6 days. Evaporation of thesolvent left 1.5 g of a residual. The crude product was purified bycolumn chromatography on silica gel (230-400 mesh, 100 mL) and elutedwith 0-5% methanol gradient in dichloromethane. This gave 0.8 g of thedesired product DLin-K-DMA. ¹H NMR (400 MHz, CDCl3) δ: 5.25-5.45 (8, m,4×CH═CH), 4.28-4.4 (1H, m, OCH), 4.1 (1H, dd, OCH), 3.53 (1H, t OCH),2.78 (4H, t, 2×C═C—CH2-C═C), 2.5-2.65 (2H, m, NCH2), 2.41 (6H, s,2×NCH3), 2.06 (8H, q, 4×allylic CH2), 1.56-1.68 (4H, m, 2×CH2),1.22-1.45 (32H, m), 0.90 (6H, t, 2×CH3) ppm.

Example 2 Synthesis of 1,2-Dilinoleyloxy-N,N-Dimethyl-3-Aminopropane(DLinDMA) DLinDMA was Synthesized as Described Below

To a suspension of NaH (95%, 5.2 g, 0.206 mol) in 120 mL of anhydrousbenzene was added dropwise N,N-dimethyl-3-aminopropane-1,2-diol (2.8 g,0.0235 mol) in 40 mL of anhydrous benzene under argon. Upon addition,the resulting mixture was stirred at room temperature for 15 min.Linoleyl methane sulfonate (99%, 20 g, 0.058 mol) in 75 mL of anhydrousbenzene was added dropwise at room temperature under argon to the abovemixture. After stirred at room temperature for 30 min., the mixture wasrefluxed overnight under argon. Upon cooling, the resulting suspensionwas treated dropwise with 250 mL of 1:1 (V:V) ethanol-benzene solution.The organic phase was washed with water (150 mL), brine (2×200 mL), anddried over anhydrous sodium sulfate. Solvent was evaporated in vacuo toafford 17.9 g of light oil as a crude product. 10.4 g of pure DLinDMAwere obtained upon purification of the crude product by columnchromatography twice on silica gel using 0-5% methanol gradient inmethylene chloride. ¹H NMR (400 MHz, CDCl3) δ: 5.35 (8H, m, CH═CH), 3.5(7H, m, OCH), 2.75 (4H, t, 2×CH2), 2.42 (2H, m, NCH2), 2.28 (6H, s,2×NCH3), 2.05 (8H, q, vinyl CH2), 1.56 (4H, m, 2×CH2), 1.28 (32H, m,16×CH2), 0.88 (6H, t, 2×CH3) ppm.

Example 3 Synthesis of2,2-Dilinoleyl-4-(2-Dimethylaminoethyl)[1,3]-Dioxolane (DLin-K-C2-DMA)

DLin-K-C2-DMA was synthesized as shown in the schematic diagram anddescription below.

Synthesis of 2,2-Dilinoleyl-4-(2-hydroxyethyl)-[1,3]-dioxolane (II)

A mixture of dilinoleyl ketone (I, previously prepared as described inExample 1, 527 mg, 1.0 mmol), 1,3,4-butanetriol (technical grade, ca.90%, 236 mg, 2 mmol) and pyridinium p-toluenesulfonate (50 mg, 0.2 mmol)in 50 mL of toluene was refluxed under nitrogen overnight with aDean-Stark tube to remove water. The resulting mixture was cooled toroom temperature. The organic phase was washed with water (2×30 mL),brine (50 mL), and dried over anhydrous Na₂SO₄. Evaporation of thesolvent resulted in a yellowish oily residual (0.6 g). The crude productwas purified by column chromatography on silica gel (230-400 mesh, 100mL) with dichloromethane as eluent. This afforded 0.5 g of pure II ascolourless oil. ¹H NMR (400 MHz, CDCl₃) δ: 5.25-5.48 (8H, m, 4×CH═CH),4.18-4.22 (1H, m, OCH), 4.08 (1H, dd, OCH), 3.82 (2H, t, OCH₂), 3.53(1H, t, OCH), 2.78 (4H, t, 2×C═C—CH₂—C═C), 2.06 (8H, q, 4×allylic CH₂),1.77-1.93 (2H, m, CH₂), 1.52-1.68 (4H, m, 2×CH₂), 1.22-1.45 (32H, m),0.86-0.94 (6H, t, 2×CH₃) ppm.

Synthesis of 2,2-Dilinoleyl-4-(2-methanesulfonylethyl)[1,3]-dioxolane(III)

To a solution of 2,2-dilinoleyl-4-(2-hydroxyethyl)-[1,3]-dioxolane (II,500 mg, 0.81 mmol) and dry triethylamine (218 mg, 2.8 mmol) in 50 mL ofanhydrous CH₂Cl₂ was added methanesulfonyl anhydride (290 mg, 1.6 mmol)under nitrogen. The resulting mixture was stirred at room temperatureovernight. The mixture was diluted with 25 mL of CH₂Cl₂. The organicphase was washed with water (2×30 mL), brine (50 mL), and dried overanhydrous Na₂SO₄. The solvent was evaporated to afford 510 mg ofyellowish oil. The crude product was used in the following step withoutfurther purification.

Synthesis of 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane(DLin-K-C2-DMA)

To the above crude material (III) under nitrogen was added 20 mL ofdimethylamine in THF (2.0 M). The resulting mixture was stirred at roomtemperature for 6 days. An oily residual was obtained upon evaporationof the solvent. Column chromatography on silica gel (230-400 mesh, 100mL) with 0-5% methanol gradient in dichloromethane as eluent resulted in380 mg of the product DLin-K-C2-DMA as pale oil. ¹H NMR (400 MHz, CDCl₃)δ: 5.27-5.49 (8, m, 4×CH═CH), 4.01-4.15 (2H, m, 2×OCH), 3.49 (1H, tOCH), 2.78 (4H, t, 2×C═C—CH₂—C═C), 2.34-2.54 (2H, m, NCH₂), 2.30 (6H, s,2×NCH₃), 2.06 (8H, q, 4×allylic CH₂), 1.67-1.95 (2H, m, CH₂), 1.54-1.65(4H, m, 2×CH₂), 1.22-1.45 (32H, m), 0.90 (6H, t, 2×CH₃) ppm.

Example 4 Synthesis of2,2-Dilinoleyl-4-(3-Dimethylaminopropyl)[1,3]-Dioxolane (DLin-K-C3-DMA)

DLin-K-C3-DMA was synthesized as described and shown in the schematicdiagram below.

Synthesis of 1,2,5-Pentanetriol

To a suspension of LiAlH₄ (1.75 g) in 80 mL of anhydrous THF was addeddropwise under nitrogen a solution of(R)-γ-hydroxymethyl-γ-butarolactone (0.50 g, 4 mmol) in 20 mL ofanhydrous THF. The resulting suspension was stirred at room temperatureunder nitrogen overnight. To this mixture was added 5.5 mL ofNaCl-saturated aqueous solution very slowly with use of an ice-waterbath. The mixture was further stirred under nitrogen overnight. Thewhite solid was filtered and washed with THF (2×20 mL). The filtrate andwashes were combined. Evaporation of the solvent gave 0.25 g ofcolourless oil as a crude product. The crude product was used in thenext step without further purification.

Synthesis of 2,2-Dilinoleyl-4-(3-hydroxypropyl)-[1,3]-dioxolane (II)

A mixture of dilinoleyl ketone (I, previously prepared as described inExample 1, 1.0 g, 2 mmol), 1,2,5-pentanetriol (crude, 0.25 g, 2 mmol)and pyridinium p-toluenesulfonate (100 mg, 0.4 mmol) in 150 mL oftoluene was refluxed under nitrogen overnight with a Dean-Stark tube toremove water. The resulting mixture was cooled to room temperature. Theorganic phase was washed with water (3×40 mL), brine (50 mL), and driedover anhydrous Na₂SO₄. Evaporation of the solvent gave a yellowish oilyresidual (1.1 g). The crude product was purified by columnchromatography on silica gel (230-400 mesh, 100 mL) with 0-1% methanolin dichloromethane as eluent. This afforded 0.90 g of pure II ascolourless oil.

Synthesis of 2,2-Dilinoleyl-4-(3-methanesulfonylpropyl)-[1,3]-dioxolane(III)

To a solution of 2,2-dilinoleyl-4-(3-hydroxypropyl)[1,3]-dioxolane (II,0.90 g, 1.4 mmol) and dry triethylamine (0.51 g, 5 mmol) in 100 mL ofanhydrous CH₂Cl₂ was added methanesulfonyl anhydride (0.70 g, 4 mmol)under nitrogen. The resulting mixture was stirred at room temperatureovernight. The organic phase was washed with water (2×40 mL), brine (50mL), and dried over anhydrous Na₂SO₄. The solvent was evaporated toafford 1.0 g of brownish oil as a crude product. The crude product wasused in the following step without further purification.

Synthesis of 2,2-Dilinoleyl-4-(3-dimethylaminopropyl)[1,3]-dioxolane(DLin-K-C3-DMA

To the above crude material (III, 1.0 g) under nitrogen was added 40 mLof dimethylamine in THF (2.0 M). The resulting mixture was stirred atroom temperature for 8 days. The solid was filtered. Upon evaporation ofthe solvent, an orange residual was resulted. Column chromatography onsilica gel (230-400 mesh, 100 mL) with 0-40% ethyl acetate gradient inhexanes as eluent resulted in 510 g of the product DLin-K-C3-DMA as paleoil. ¹H NMR (400 MHz, CDCl₃) δ: 5.22-5.50 (8, m, 4×CH═CH), 3.95-4.15(2H, m, 2×OCH), 3.35-3.55 (1H, m OCH), 2.78 (4H, t, 2×C═C—CH₂—C═C),2.45-2.55 (2H, m, NCH₂), 2.35 (6H, s, 2×NCH₃), 2.05 (8H, q, 4×allylicCH₂), 1.45-1.75 (6H, m, CH₂), 1.2-1.45 (32H, m), 0.90 (6H, t, 2×CH₃)ppm.

Example 5 Synthesis of2,2-Dilinoleyl-4-(4-Dimethylaminobutyl)-[1,3]-Dioxolane (DLin-K-C4-DMA)

DLin-K-C4-DMA was synthesized as described and shown in the schematicdiagram below.

Synthesis of 2,2-Dilinoleyl-4-(4-hydroxybutyl)-[1,3]-dioxolane (II)

A mixture of dilinoleyl ketone (I, previously prepared as described inExample 1, 1.05 g, 2.0 mmol), 1,2,6-hexanetriol (0.54 g, 4 mmol) andpyridinium p-toluenesulfonate (100 mg, 0.4 mmol) in 150 mL of toluenewas refluxed under nitrogen overnight with a Dean-Stark tube to removewater. The resulting mixture was cooled to room temperature. The organicphase was washed with water (2×60 mL), brine (60 mL), and dried overanhydrous Na₂SO₄. Evaporation of the solvent resulted in a yellowishoily residual (1.5 g). The crude product was purified by columnchromatography on silica gel (230-400 mesh, 100 mL) with 0-0.5% methanolin dichloromethane as eluent. This afforded 1.4 g of pure II ascolourless oil.

Synthesis of 2,2-Dilinoleyl-4-(4-methanesulfonylbutyl)-[1,3]-dioxolane

To a solution of 2,2-dilinoleyl-4-(4-hydroxybutyl)[1,3]-dioxolane (H,1.4 g, 2 mmol) and dry triethylamine (0.73 g, 7.2 mmol) in 150 mL ofanhydrous CH₂Cl₂ was added methanesulfonyl anhydride (1.0 g, 5.7 mmol)under nitrogen. The resulting mixture was stirred at room temperatureovernight. The organic phase was washed with water (2×75 mL), brine (75mL), and dried over anhydrous Na₂SO₄. The solvent was evaporated toafford 1.45 g of pale oil as a crude product. The crude product was usedin the following step without further purification.

Synthesis of 2,2-Dilinoleyl-4-(4-dimethylaminobutyl)[1,3]-dioxolane(DLin-K-C4-DMA

To the above crude material (III, 1.45 g) under nitrogen was added 60 mLof dimethylamine in THF (2.0 M). The resulting mixture was stirred atroom temperature for 6 days. The solid was filtered. An oily residual(1.2 g) was obtained upon evaporation of the solvent. Columnchromatography on silica gel (230-400 mesh, 100 mL) with 0-5% methanolgradient in dichloromethane as eluent resulted in 0.95 g of the productDLin-K-C4-DMA as pale oil. ¹H NMR (400 MHz, CDCl₃) δ: 5.26-5.49 (8, m,4×CH═CH), 3.97-4.15 (2H, m, 2×OCH), 3.45 (1H, t OCH), 2.78 (4H, t,2×C═C—CH₂—C═C), 2.45-2.55 (2H, m, NCH₂), 2.40 (6H, s, 2×NCH₃), 2.05 (8H,q, 4×allylic CH₂), 1.45-1.75 (8H, m, CH₂), 1.2-1.45 (32H, m), 0.90 (6H,t, 2×CH₃) ppm.

Example 6 Synthesis of2,2-Dilinoleyl-5-Dimethylaminomethyl-[1,3]-Dioxane (DLin-K6-DMA)

DLin-K6-DMA was synthesized as described and shown in the schematicdiagram below.

Synthesis of 2,2-Dilinoleyl-5-hydroxymethyl)[1,3]-dioxane (II)

A mixture of dilinoleyl ketone (I, previously prepared as described inExample 1, 1.05 g, 2.0 mmol), 2-hydroxymethyl-1,3-propanediol (475 mg, 4mmol) and pyridinium p-toluenesulfonate (100 mg, 0.4 mmol) in 150 mL oftoluene was refluxed under nitrogen overnight with a Dean-Stark tube toremove water. The resulting mixture was cooled to room temperature. Theorganic phase was washed with water (2×60 mL), brine (60 mL), and driedover anhydrous Na₂SO₄. Evaporation of the solvent resulted in pale oil(1.2 g). The crude product was purified by column chromatography onsilica gel (230-400 mesh, 100 mL) with 0-1% methanol gradient indichloromethane as eluent. This afforded 1.0 g of pure II as colourlessoil.

Synthesis of 2,2-Dilinoleyl-5-methanesulfonylmethyl-[1,3]-dioxane (III)

To a solution of 2,2-dilinoleyl-5-hydroxymethyl-[1,3]-dioxane (II, 1.0g, 1.6 mmol) and dry triethylamine (430 mg, 4.2 mmol) in 120 mL ofanhydrous CH₂Cl₂ was added methanesulfonyl anhydride (600 mg, 3.3 mmol)under nitrogen. The resulting mixture was stirred at room temperatureovernight. The organic phase was washed with water (2×60 mL), brine (60mL), and dried over anhydrous Na₂SO₄. The solvent was evaporated toafford 1.1 g of pale oil. The crude product was used in the followingstep without further purification.

Synthesis of 2,2-Dilinoleyl-5-dimethylaminomethyl)-[1,3]-dioxane(DLin-K6-DMA)

To the above crude material (III, 1.1 g) under nitrogen was added 20 mLof dimethylamine in THF (2.0 M). The resulting mixture was stirred atroom temperature for 7 days. An oily residual was obtained uponevaporation of the solvent. Column chromatography on silica gel (230-400mesh, 100 mL) with 0-30% ethyl acetate gradient in hexanes as eluentresulted in 260 mg of the product DLin-K⁶-DMA as pale oil. ¹H NMR (400MHz, CDCl₃) δ: 5.24-5.51 (8, m, 4×CH═CH), 4.04 (2H, dd, 2×OCH)), 3.75(2H, dd OCH), 2.7-2.9 (2H, br, NCH₂), 2.78 (4H, t, 2×C═C—CH₂—C═C), 2.57(6H, s, 2×NCH₃), 1.95-2.17 (9H, q, 4×allylic CH₂ and CH), 1.67-1.95 (2H,m, CH₂), 1.54-1.65 (4H, m, 2×CH₂), 1.22-1.45 (32H, m), 0.90 (6H, t,2×CH₃) ppm.

Example 7 Synthesis of Dilinoleylmethyl 3-Dimethylaminopropionate(DLin-M-K-DMA)

DLin-M-K-DMA was synthesized as described and shown in the schematicdiagram below.

Synthesis of Dilinoleylmethanol (II)

To a solution of dilinoley ketone (I, 1.3 g) in methanol (130 mL) wasadded NaBH₄ (0.7 g). The resulting solution was stirred at roomtemperature for 60 min. The mixture was poured into 300 mL of ice water.The aqueous phase was extracted with ether (3×100 mL). The combinedether phase was washed with water (100 mL), brine (100 mL) and driedover anhydrous Na₂SO₄. Evaporation of the solvent gave a yellowish oilyresidual (1.4 g). The crude product was purified by columnchromatography on silica gel (230-400 mesh, 100 mL) with 0-5% ethylacetate gradient in hexanes as eluent. This resulted in 1.1 g of pure IIas pale oil.

Synthesis of Dilinoleylmethyl 3-Bromopropionate (III)

To a solution of dilinoleylmethanol (II, 560 mg, 1 mmol) and drytriethylamine (0.44 g, 4.2 mmol) in 50 mL of anhydrous CH₂Cl₂ was addeddropwise 3-bromopropionyl chloride (technical grade, 0.34 mL) undernitrogen. The resulting mixture was stirred at room temperature for 3days. The organic phase was diluted with 50 mL of dichloromethane andwashed with water (3×50 mL), brine (50 mL), and dried over anhydrousNa₂SO₄. The solvent was evaporated to afford 610 mg of brownish oil as acrude product. The crude product was purified by column chromatographyon silica gel (230-400 mesh, 100 mL) with 0-3% ethyl acetate gradient inhexanes as eluent. This resulted in 540 g of a mixture of III as a majorproduct and a by-product. The mixture was used in the following stepwithout further purification.

Synthesis of Dilinoleylmethyl 3-Dimethylaminopropionate (DLin-M-K-DMA)

To the above mixture (III, 540 mg) under nitrogen was added 15 mL ofdimethylamine in THF (2.0 M). The resulting mixture was stirred at roomtemperature for 8 days. The solid was filtered. Upon evaporation of thesolvent, a brownish residual was resulted. Column chromatography onsilica gel (230-400 mesh, 100 mL) with 0-3% methanol in dichloromethaneas eluent resulted in 430 mg of the product DLin-M-K-DMA as pale oil. ¹HNMR (400 MHz, CDCl₃) δ: 5.25-5.50 (8, m, 4×CH═CH), 4.70-5.00 (1H, q,OCH), 2.8-3.0 (2H, m, NCH₂), 2.78 (4H, t, 2×C═C—CH₂—C═C), 2.6-2.7 (2H,m, COCH₂), 2.45 (6H, s, 2×NCH₃), 2.05 (8H, q, 4×allylic CH₂), 1.45-1.75(4H, m, CH₂), 1.2-1.45 (32H, m), 0.90 (6H, t, 2×CH₃) ppm.

Example 8 Synthesis of2,2-Dilinoleyl-4-N-Methylpepiazino-[1,3]-Dioxolane (DLin-K-MPZ)

DLin-K-MPZ was synthesized as described below and shown in the followingdiagram.

Step 1

To a mixture of dilinoleyl ketone (I, 1.3 gm, 2.5 mmol),3-Bromo1,2-propane diol (1.5 gm, 9.7 mmol) and PPTS(Pyridinium-p-toluene sulfonate) (100 mg) in 25 mL of Toluene wasrefluxed under nitrogen for over night with a Dean-stark tube to removewater. The resulting mixture was cooled to room temperature. The organicphase was washed with water (2×50 mL) and saturated NaHCO₃ solution,dried over anhydrous Na₂SO₄, evaporation of solvent resulted in ayellowish oily residue. Column Chromatography on silica (230-400 mesh),with 0-5% ether as eluent in hexanes afforded 750 mg of the ketal, whichwas further reacted with Methyl piperzine as follows.

Step 2

To a mixture of D-Lin-Ketal (II, 250 mg, 0.37 mmol) and K₂CO₃ (138 mg, 1mmol) in 5 mL of acetonitrile was added Morpholine (50 mg, 0.50 mmol).Then the resulting solution was refluxed under argon overnight. Theresulting mixture was cooled to room temperature, solvent was evaporatedthe organic phase was washed with water (2×50 mL), and dried overanhydrous Na₂SO₄. Evaporation of the solvent resulted in yellowish oilyresidue. Column chromatography on silica gel (230-400 mesh, 500 mL)eluted with 25-50% hexanes and ethyl acetate, and then eluted with 0-5%methanol gradient in dichloromethane. This gave 225 mg of the desiredproduct D-Lin-K-N-methylpiperzine(D-Lin-K-MPZ).

¹H NMR (300 MHz, CDCl₃) δ: 5.27-5.46 (8H, m), 4.21-4.31 (1H, m),4.06-4.09 (1H, t), 3.49-3.57 (1H, t) 3.49-3.55 (1H, t), 2.75-2.81 (4H,t) 2.42-2.62 (8H, m), 2.30 (3H, s), 2.02-2.09 (8H, m) 1.55-1.65 (4H, m),1.2-1.47 (32H, m), 0.87-0.90 (6H, t) ppm.

Example 9 Synthesis of2,2-Dioleoyl-4-Dimethylaminomethyl-[1,3]-Dioxolane (DO-K-DMA)

DO-K-DMA having the structure shown below was prepared using a methodsimilar to the method described in Example 1 for producing D-Lin-K-DMA,except the initial starting material was oleoyl methane sulfonate,instead of linoleyl methane sulfonate.

¹H NMR (300 MHz, CDCl₃) δ: 5.32-5.40 (4H, m), 4.21-4.31 (1H, m),4.06-4.10 (1H, t), 3.49-3.55 (1H, t), 2.5-2.6 (2H, m), 2.35 (6H, s),1.90-2.00 (8H, m), 1.70-1.80 (2H, m), 1.55-1.65 (8H, m), 1.2-1.47 (40H,m), 0.87-0.90 (6H, t) ppm.

Example 10 Synthesis of2,2-Distearoyl-4-Dimethylaminomethyl-[1,3]-Dioxolane (DS-K-DMA)

DS-K-DMA having the structure shown below was synthesized as describedbelow.

To a solution of DO-K-DMA prepared as described in Example 8 (250 mg,0.4 mmol) was added in ethanol Palladium charcoal, and the resultingmixture was stirred under hydrogen atmosphere over night. The reactionmixture was filtered through celite, the solvent was evaporated, andthen the crude product was purified by column chromatography on silicagel (230-400 mesh, 500 mL) and eluted with 25-50% ethyl acetate asgradient in hexanes. This gave white solid 225 mg of the desired productDS-K-DMA.

¹H NMR (300 MHz, CDCl₃) δ: 4.21-4.31 (1H, m), 4.06-4.09 (1H, t),3.49-3.55 (1H, t), 2.5-2.6 (2H, m), 2.35 (6H, s), 1.55-1.65 (4H, m),1.2-1.47 (40H, m), 0.87-0.90 (6H, t) ppm.

Example 11 Synthesis of 2,2-Dilinoleyl-4-N-Morpholino[1,3]-Dioxolane(DLin-K-MA)

Dlin-K-MA having the structure shown below was synthesized as describedbelow.

To a mixture of D-Lin-Ketal (I, 250 mg, 0.37 mmol) and K₂CO₃ (138 mg, 1mmol) in 5 mL of acetonitrile was added Morpholine (50 mg, 0.57 mmol).Then the resulting solution was refluxed under argon overnight. Theresulting mixture was cooled to room temperature, solvent wasevaporated, the organic phase was washed with water (2×50 mL), and driedover anhydrous Na₂SO₄. Evaporation of the solvent resulted in yellowishoily residue.

Column chromatography on silicagel (230-400 mesh, 500 mL) eluted with25-50% hexanes and ethyl acetate, and then eluted with 0-5% methanol asgradient in dichloromethane. This gave 225 mg of the desired productDLin-K-MA.

¹H NMR (300 MHz, CDCl₃) δ: 5.27-5.46 (8H, m), 4.21-4.31 (1H, m),4.06-4.09 (1H, t), 3.71-3.73 (4H, t) 3.49-3.55 (1H, t), 2.78 (4H, t)2.42-2.62 (6H, m), 2.02-2.09 (8H, m) 1.55-1.65 (4H, m), 1.2-1.47 (32H,m), 0.87-0.90 (6H, t) ppm.

Example 12 Synthesis of 2,2-Dilinoleyl-4-Trimethylamino-[1,3]-DioxolaneChloride (DLin-K-TMA.CL)

DLin-K-TMA.Cl was Synthesized as Described and Shown in the schematicdiagram below.

Synthesis of 2,2-Dilinoleyl-4-dimethylamino-[1,3]-dioxolane (DLin-K-DMA)

DLin-K-DMA was prepared as described in Example 1.

Synthesis of 2,2-Dilinoleyl-4-trimethylamino-[1,3]-dioxolane Chloride(DLin-K-TMA.I)

A mixture of 2,2-dilinoleyl-4-dimethylamino-[1,3]-dioxolane (DLin-K-DMA,1.5 g, 2.4 mmol) and CH₃I (4.0 mL, 64 mmol) in 10 mL of anhydrous CH₂Cl₂was stirred under nitrogen at room temperature for 9 days. Evaporationof the solvent and excess of iodomethane afforded 20 g of yellow syrupas crude DLin-K-TMA.I, which was used in the following step withoutfurther purification.

Preparation of 2,2-Dilinoleyl-4-trimethylamino-[1,3]-dioxolane Chloride(DLin-K-TMA.Cl)

The above crude DLin-K-TMA.I (2.0 g) was dissolved in 100 mL of CH₂Cl₂in a separatory funnel. 30 mL of 1N HCl methanol solution was added, andthe resulting solution was shaken well. To the solution was added 50 mLof brine and the mixture was shaken well. The organic phase wasseparated. The aqueous phase was extracted with 10 mL of CH₂Cl₂. Theorganic phase and extract were then combined. This completed the firststep of ion exchange. The ion exchange step was repeated four moretimes. The final organic phase was washed with brine (2×75 mL) and driedover anhydrous Na₂SO₄. Evaporation of the solvent gave 2.0 g ofyellowish viscous oil. The product was purified by column chromatographyon silica gel (230-400 mesh, 100 mL) eluted with 0-15% methanol gradientin chloroform. This afforded 1.2 g of2,2-dilinoleyl-4-trimethylamino-[1,3]-dioxolane chloride (DLin-K-TMA.Cl)as a pale waxy material. ¹H NMR (300 MHz, CDCl₃) δ: 5.25-5.45 (8H, m,4×CH═CH), 4.55-4.75 (2H, m, 2×OCH), 4.26-4.38 (1H, dd, OCH), 3.48-3.57(1H, dd, NCH), 3.51 (9H, s, 3×NCH₃), 3.11-3.22 (1H, dd, NCH), 2.77 (4H,t, 2×C═C—CH₂—C═C), 2.05 (8H, q, 4×allylic CH₂), 1.49-1.7 (4H, m, 2×CH₂),1.2-1.45 (30H, m), 0.89 (6H, t, 2×CH₃) ppm.

Example 13 Synthesis of 2,2-Dilinoleyl-4,5-Bis(DimethylaminoMethyl)-[1,3]-Dioxolane (DLin-K²-DMA)

DLin-K²-DMA was synthesized as described and shown in the schematicdiagrams below.

Synthesis of D-Lin-K-diethyltartarate (II)

A mixture of D-Lin-Ketone (I, 1 gram, 1.9 mmol), Diethyl-D-tartarate(412 mg, 2 mmol) and Pyridinium p-tolene sulfonate (250 mg, 1 mmol) in25 mL of toluene was refluxed under nitrogen for two days with aDean-stark tube to remove water. The resulting mixture was cooled toroom temperature. The organic phase was washed with water NaHCO₃ andbrine (2×50 mL) and dried over anhydrous Na₂SO₄. Evaporation of thesolvent resulted in yellowish oily residue. Column chromatography onsilica gel (230-400 mesh, 500 mL) eluted with 0-10% ether gradients inhexanes as eluent afforded 400 mg of pure D-Lin-diethyltartarate (II).

¹H NMR (300 MHz, CDCl₃) δ: 5.27-5.46 (8H, m), 4.67 (2H, s), 4.20-4.30(1H, t), 2.75 (4H, t), 2.02-2.09 (8H, m) 1.62-1.72 (4H, m), 1.2-1.47(32H, m), 0.87-0.90 (6H, t) ppm.

Synthesis of D-Lin-K-diethyldiol (III)

To a solution of Lithiumaluminumhydride (32 mg, 1 mmol) in dry THF asolution of D-Lin-K-diethyltartarate (II, 600 mg, 0.85 m mol) was addedin dry THF at 0° C. under argon atmosphere and then the reaction wasstirred for four hours at room temperature. The reaction mixture wasquenched with ice cold water and then filtered through celite and theevaporation of solvent gave crude reduced alcohol. Column chromatographyon silica gel (230-400 mesh, 500 mL) eluted with 10-40% ethyl acetategradients in hexanes as eluent afforded 350 mg of pureD-Lin-diethyltartarate (III).

¹H NMR (300 MHz, CDCl₃) δ: 5.27-5.46 (8H, m), 3.95 (2H, t), 3.65-3.85(4H, dd), 2.75 (4H, t), 2.02-2.09 (8H, m) 1.62-1.72 (4H, m), 1.2-1.47(32H, m), 0.87-0.90 (6H, t) ppm.

Synthesis of D-Lin-K-diethyldimesylate (IV)

To a mixture of D-Lin-K-diethyltartarate (III) alcohol (570 mg, 0.95mmol) in dry dichloromethane pyridine (275 mg, 3.85 mmol) and4-(Dimethylamino)pyridine (122 mg, 1 mmol) was added under argonatmosphere to this solution a solution of methane sulfonyl chloride (500mg, 2.5 mmol) was slowly added and stirred over night.

The organic phase was washed with water and brine (2×50 mL) then solventwas evaporated to give yellowish oil residue. Purified over Columnchromatography on silica gel (230-400 mesh, 500 mL), eluted with 10-40%ethyl acetate gradients in hexanes as eluent, afforded 300 mg of pureD-Lin-diethyltartarate (IV).

¹H NMR (300 MHz, CDCl₃) δ: 5.27-5.46 (8H, m), 4.35 (4H, d), 4.12-4.17(2H, t), 3.08 (6H, s), 2.75 (4H, t), 2.02-2.09 (8H, m) 1.62-1.72 (4H,m), 1.2-1.47 (32H, m), 0.87-0.90 (6H, t) ppm.

Synthesis of D-Lin-K²-DMA

Anhydrous dimethyl amine solution in THF was added to the reactionvessel containing (300 mg) of D-Lin-diethyltartarate (IV) at roomtemperature for 5 min. the reaction flask was then sealed and themixture stirred at room temperature for 6 days. Evaporation of thesolvent left 300 mg of residual. the crude product was purified bycolumn chromatography on silica gel (230-400 mesh, 500 mL) eluted with0-10% Methanol gradients in chloroform as eluent afforded 50 mg of pureD-Lin-K²-DMA.

¹H NMR (300 MHz, CDCl₃) δ: 5.27-5.46 (8H, m), 3.72-3.80 (2H, t), 2.75(4H, t), 2.49 (4H, d), 2.30 (12H, s), 2.02-2.09 (8H, m) 1.62-1.72 (4H,m), 1.2-1.47 (32H, m), 0.87-0.90 (6H, t) ppm.

Example 14 Synthesis of D-Lin-K-N-Methylpiperzine

D-Lin-K-N-methylpiperzine having the structure shown below was preparedas described below.

To a mixture of D-Lin-Ketal (I, 250 mg, 0.37 mmol) and K₂CO₃ (138 mg, 1mmol) in 5 mL of acetonitrile was added Morpholine (50 mg, 0.50 mmol).Then the resulting solution was refluxed under argon overnight. Theresulting mixture was cooled to room temperature, solvent was evaporatedthe organic phase was washed with water (2×50 mL), and dried overanhydrous Na₂SO₄. Evaporation of the solvent resulted in yellowish oilyresidue. Column chromatography on silica gel (230-400 mesh, 500 mL),eluted with 25-50% hexanes and ethyl acetate, and then eluted with 0-5%methanol gradient in dichloromethane. This gave 225 mg of the desiredproduct D-Lin-K-N-methylpiperzine.

¹H NMR (300 MHz, CDCl₃) δ: 5.27-5.46 (8H, m), 4.21-4.31 (1H, m),4.06-4.09 (1H, t), 3.49-3.57 (1H, t) 3.49-3.55 (1H, t), 2.75-2.81 (4H,t) 2.42-2.62 (8H, m), 2.30 (3H, s), 2.02-2.09 (8H, m) 1.55-1.65 (4H, m),1.2-1.47 (32H, m), 0.87-0.90 (6H, t) ppm.

Example 15 Synthesis of mPEG2000-1,2-Di-O-Alkyl-SN3-Carbomoylglyceride(PEG-C-DOMG)

The PEG-lipids, such as mPEG2000-1,2-Di-O-Alkyl-sn3-Carbomoylglyceride(PEG-C-DOMG) were synthesized as shown in the schematic and describedbelow.

Synthesis of IVa

1,2-Di-O-tetradecyl-sn-glyceride Ia (30 g, 61.80 mmol) andN,N′-succinimidylcarboante (DSC, 23.76 g, 1.5 eq) were taken indichloromethane (DCM, 500 mL) and stirred over an ice water mixture.Triethylamine (TEA, 25.30 mL, 3 eq) was added to the stirring solutionand subsequently the reaction mixture was allowed to stir overnight atambient temperature. Progress of the reaction was monitored by TLC. Thereaction mixture was diluted with DCM (400 mL) and the organic layer waswashed with water (2×500 mL), aqueous NaHCO₃ solution (500 mL) followedby standard work-up. The residue obtained was dried at ambienttemperature under high vacuum overnight. After drying, the crudecarbonate IIa thus obtained was dissolved in dichloromethane (500 mL)and stirred over an ice bath. To the stirring solution, mPEG₂₀₀₀-NH₂(III, 103.00 g, 47.20 mmol, purchased from NOF Corporation, Japan) andanhydrous pyridine (Py, 80 mL, excess) were added under argon. In someembodiments, the x in compound III has a value of 45-49, preferably47-49, and more preferably 49. The reaction mixture was then allowed tostir at ambient temperature overnight. Solvents and volatiles wereremoved under vacuum and the residue was dissolved in DCM (200 mL) andcharged on a column of silica gel packed in ethyl acetate. The columnwas initially eluted with ethyl acetate and subsequently with gradientof 5-10% methanol in dichloromethane to afford the desired PEG-Lipid IVaas a white solid (105.30 g, 83%). ¹H NMR (CDCl₃, 400 MHz) δ=5.20-5.12(m, 1H), 4.18-4.01 (m, 2H), 3.80-3.70 (m, 2H), 3.70-3.20 (m,—O—CH₂—CH₂—O—, PEG-CH₂), 2.10-2.01 (m, 2H), 1.70-1.60 (m, 2H), 1.56-1.45(m, 4H), 1.31-1.15 (m, 48H), 0.84 (t, J=6.5 Hz, 6H). MS range found:2660-2836.

Synthesis of IVb

1,2-Di-O-hexadecyl-sn-glyceride Ib (1.00 g, 1.848 mmol) and DSC (0.710g, 1.5 eq) were taken together in dichloromethane (20 mL) and cooleddown to 0° C. in an ice water mixture. Triethylamine (1.00 mL, 3 eq) wasadded and the reaction was stirred overnight. The reaction was followedby TLC, diluted with DCM, washed with water (2 times), NaHCO₃ solutionand dried over sodium sulfate. Solvents were removed under reducedpressure and the resulting residue of IIb was maintained under highvacuum overnight. This compound was directly used for the next reactionwithout further purification. MPEG₂₀₀₀-NH₂ III (1.50 g, 0.687 mmol,purchased from NOF Corporation, Japan) and IIb (0.702 g, 1.5 eq) weredissolved in dichloromethane (20 mL) under argon. In some embodiments,the x in compound III has a value of 45-49, preferably 47-49, and morepreferably 49. The reaction was cooled to 0° C. Pyridine (1 mL, excess)was added and the reaction stirred overnight. The reaction was monitoredby TLC. Solvents and volatiles were removed under vacuum and the residuewas purified by chromatography (first ethyl acetate followed by 5-10%MeOH/DCM as a gradient elution) to obtain the required compound IVb as awhite solid (1.46 g, 76%). ¹H NMR (CDCl₃, 400 MHz) δ=5.17 (t, J=5.5 Hz,1H), 4.13 (dd, J=4.00 Hz, 11.00 Hz, 1H), 4.05 (dd, J=5.00 Hz, 11.00 Hz,1H), 3.82-3.75 (m, 2H), 3.70-3.20 (m, —O—CH₂—CH₂—O—, PEG-CH₂), 2.05-1.90(m, 2H), 1.80-1.70 (m, 2H), 1.61-1.45 (m, 6H), 1.35-1.17 (m, 56H), 0.85(t, J=6.5 Hz, 6H). MS range found: 2716-2892.

Synthesis of IVc

1,2-Di-O-octadecyl-sn-glyceride Ic (4.00 g, 6.70 mmol) and DSC (2.58 g,1.5 eq) were taken together in dichloromethane (60 mL) and cooled downto 0° C. in an ice water mixture. Triethylamine (2.75 mL, 3 eq) wasadded and the reaction was stirred overnight. The reaction was followedby TLC, diluted with DCM, washed with water (2 times), NaHCO₃ solution,and dried over sodium sulfate. Solvents were removed under reducedpressure and the residue was maintained under high vacuum overnight.This compound was directly used for the next reaction without furtherpurification. MPEG₂₀₀₀-NH₂ III (1.50 g, 0.687 mmol, purchased from NOFCorporation, Japan) and IIc (0.760 g, 1.5 eq) were dissolved indichloromethane (20 mL) under argon. In some embodiments, the x incompound III has a value of 45-49, preferably 47-49, and more preferably49. The reaction was cooled to 0° C. Pyridine (1 mL, excess) was addedand the reaction was stirred overnight. The reaction was monitored byTLC. Solvents and volatiles were removed under vacuum and the residuewas purified by chromatography (ethyl acetate followed by 5-10% MeOH/DCMas a gradient elution) to obtain the desired compound IVc as a whitesolid (0.92 g, 48%). ¹H NMR (CDCl₃, 400 MHz) δ=5.22-5.15 (m, 1H), 4.16(dd, J=4.00 Hz, 11.00 Hz, 1H), 4.06 (dd, J=5.00 Hz, 11.00 Hz, 1H),3.81-3.75 (m, 2H), 3.70-3.20 (m, —O—CH₂—CH₂—O—, PEG-CH₂), 1.80-1.70 (m,2H), 1.60-1.48 (m, 4H), 1.31-1.15 (m, 64H), 0.85 (t, J=6.5 Hz, 6H). MSrange found: 2774-2948.

Example 16 Influence of Cationic Lipid on In Vivo Gene Silencing

It is well established that in vivo RNAi silencing of specifichepatocyte proteins can be achieved following intravenous (i.v.)administration of siRNA's encapsulated in, or associated with, selectnanoparticles designed for intracellular delivery. One of the mostactive, and well characterized of these is a stable nucleic acid lipidparticle (SNALP) containing the cationic lipid1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA). In this Example,rational design in combination with in vivo screening were applied tosystematically modify the structure of DLinDMA and identify molecularfeatures that enhance or reduce cationic lipid potency. More than 30lipids were synthesized and incorporated into nucleic acid-lipidparticles, i.e., lipid nanoparticles (LN) encapsulating an siRNA(LN-siRNA) targeting Factor VII (FVII), a blood clotting componentsynthesized and secreted by hepatocytes that is readily measured inserum. LN-siRNA systems were prepared using the same process, lipidmolar ratios and particle size to minimize effects on activity resultingfrom formulation characteristics other than the cationic lipid. Eachformulation was administered as a single bolus injection over a range ofdoses enabling an estimate of the siRNA dose required to reduce FVIIserum protein concentrations by 50% after 24 h (ED₅₀).

The studies described herein were performed using the followingmaterials and methods.

Materials and Methods

Lipids

Cationic lipids were synthesized as described in the previous Examples.Distearoylphosphatidylcholine (DSPC) was purchased from Northern Lipids(Vancouver, Canada). Cholesterol was purchased from Sigma ChemicalCompany (St. Louis, Mo., USA) or Solvay Pharmaceuticals (Weesp, TheNetherlands).

The synthesis of N-[(methoxy poly(ethyleneglycol)₂₀₀₀)carbamyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-C-DMA) andN-[(methoxy poly(ethyleneglycol)₂₀₀₀)succinimidyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-S-DMA)were as described by Hayes et. al., J. Control Release 112:280-290(2006). R-3-[(w-methoxy-poly(ethyleneglycol)2000)carbamoyl)]-1,2-dimyristyloxlpropyl-3-amine (PEG-C-DOMG) wassynthesized as described herein and by Akinc et. al., Nat. Biotechnol.26:561-56 (2008). These three PEG-lipids were interchangeable in theformulation without impacting activity (data not shown), Therefore,throughout the text, they are referred to generally as PEG-lipid forpurposes of clarity.

Synthesis of siRNA

All siRNAs and 2′-OMe oligoribonucleotides were synthesized by Alnylamas described in John et al. (Nature advance online publication, 26 Sep.2007 (D01:10.1038/nature06179)). Oligonucleotides were characterized byelectrospray mass spectrometry and anion exchange HPLC.

Sequences of siRNAs used in these studies were as follows:

(SEQ ID NO: 33) si-FVII sense, 5′ GGAUCAUCUCAAGUCUUACTT 3′;(SEQ ID NO: 34) si-FVII antisense, 5′-GUAAGACUUGAGAUGAUCCTT-3′;(SEQ ID NO: 35)  si-Luc sense, 5′-cuuAcGcuGAGuAcuucGATT-3′;(SEQ ID NO: 36) si-Luc antisense, 5′-UCGAAGuACUcAGCGuAAGTT-3′,with lower-case letters denoting 2′-O-Me-modified nucleotides; andunderlined letters denoting 2′-F-modified nucleotides. All siRNAscontained phosphorothioate linkages between the two thymidines (T) atthe 3′ end of each strand.

Preformed Vesicle Method to Formulate Nucleic Acid-Lipid Particles

Nucleic acid-lipid particles were made using the preformed vesicle (PFV)method, essentially as described in Maurer et al. (Biophys J., 2001).Cationic lipid, DSPC, cholesterol and PEG-lipid were solubilised inethanol at a molar ratio of 40/10/40/10, respectively. The lipid mixturewas added to an aqueous buffer (50 mM citrate, pH 4) with mixing to afinal ethanol and lipid concentration of 30% (vol/vol) and 6.1 mg/mLrespectively and allowed to equilibrate at room temperature for 2 minbefore extrusion. The hydrated lipids were extruded through two stacked80 nm pore-sized filters (Nuclepore) at 22° C. using a Lipex Extruder(Hope, M. J. et al. Biochim. Biophys. Acta 812:55-65 (1985)) until avesicle diameter of 70-90 nm, as determined by Nicomp analysis, wasobtained. This generally required 1-3 passes. The FVII siRNA(solubilised in a 50 mM citrate, pH 4 aqueous solution containing 30%ethanol) was added to the pre-equilibrated (35° C.) vesicles, at a rateof ˜5 mL/min with mixing. After a final target siRNA/lipid ratio of 0.06(wt/wt) was achieved, the mixture was incubated for a further 30 min at35° C. to allow vesicle re-organization and encapsulation of the FVIIsiRNA. The ethanol was then removed and the external buffer replacedwith PBS (155 mM NaCl, 3 mM Na2HPO4, 1 mM KH2PO4, pH 7.5) by eitherdialysis or tangential flow diafiltration.

Particle Size Analysis

The size distribution of liposomal siRNA formulations was determinedusing a NICOMP Model 380 Sub-micron particle sizer (PSS NICOMP, ParticleSizing Systems, Santa Barbara, Calif.). Mean particle diameters weregenerally in the range 50-120 nm, depending on the lipid compositionused. Liposomal siRNA formulations were generally homogeneous and hadstandard deviations (from the mean particle size) of 20-50 nm, dependingon the lipid composition and formulation conditions used.

Measurement of Free siRNA by Ion Exchange Chromatography

Anion exchange chromatography using either DEAE Sepharose columns orcommercial centrifugal devices (Vivapure D Mini columns) was used tomeasure the amount of free siRNA in the sample. For the DEAE Sepharosecolumns, siRNA-containing formulations were eluted through the columns(˜2.5 cm bed height, 1.5 cm diameter) equilibrated with HBS (145 mMNaCl, 20 mM HEPES, pH 7.5). An aliquot of the initial and eluted samplewere assayed for lipid and siRNA content by HPLC and A260, respectively.The percent encapsulation was calculated based on the change in siRNA tolipid ratios between the pre and post column samples. For the Vivapurecentrifugal devices, an aliquot (0.4 mL, <1.5 mg/mL siRNA) of thesiRNA-containing formulation was eluted through the positively chargedmembrane by centrifugation (2000×g for 5 min). Aliquots of the pre andpost column samples were analyzed as described above to determine theamount of free siRNA in the sample.

Determination of siRNA Concentration

siRNA concentration was determined by measuring the absorbance at 260 nmafter solubilization of the lipid. The lipid was solubilized accordingto the procedure outlined by Bligh and Dyer (Bligh, et al., Can. J.Biochem. Physiol. 37:911-917 (1959). Briefly, samples of liposomal siRNAformulations were mixed with chloroform/methanol at a volume ratio of1:2.1:1 (aqueous sample:methanol:chloroform). If the solution was notcompletely clear (i.e., a single, clear phase) after mixing, anadditional 50-100 mL (volume recorded) of methanol was added and thesample was remixed. Once a clear monophase was obtained, the sample wasassayed at 260 nm using a spectrophotometer. siRNA concentration wasdetermined from the A260 readings using a conversion factor ofapproximately 45 μg/mL=1.0 OD, using a 1.0 cm path length. Theconversion factor in the chloroform/methanol/water monophase varies(35-50 μg/mL=1.0 OD) for each lipid composition and is determinedempirically for each novel lipid formulation using a known amount ofsiRNA.

Determination of Lipid Concentrations and Ratios

Cholesterol, DSPC, PEG-lipid, and the various cationic lipids weremeasured against reference standards using a Waters Alliance HPLC systemconsisting of an Alliance 2695 Separations Module (autosampler, HPLCpump, and column heater), a Waters 2424 Evaporative Light ScatteringDetector (ELSD), and Waters Empower HPLC software (version 5.00.00.00,build number 1154; Waters Corporation, Milford, Mass., USA). Samples (15μL) containing 0.8 mg/mL total lipid in 90% ethanol were injected onto areversed-phase XBridge C18 column with 2.5 μm packing, 2.1 mm×50 mm(Waters Corporation, Milford, Mass., USA) heated at 55° C. andchromatographed with gradient elution at a constant flow rate of 0.5mL/min. The mobile phase composition changed from 10 mM NH₄HCO₃:methanol(20:80) to THF:10 mM NH₄HCO₃:methanol (16:4:80) over 16 minutes. The gaspressure on the ELSD was set at 25 psi, while the nebulizerheater-cooler set point and drift tube temperature set point were set at100% and 85° C. respectively. Measured lipid concentrations (mg/mL) wereconverted to molar concentrations, and relative lipid ratios wereexpressed as mol % of the total lipid in the formulation.

Determination of Encapsulation Efficiency

Trapping efficiencies were determined after removal of external siRNA bytangential flow diafiltration or anion exchange chromatography. siRNAand lipid concentrations were determined (as described above) in theinitial formulation incubation mixtures and after tangential flowdiafiltration. The siRNA-to-lipid ratio (wt/wt) was determined at bothpoints in the process, and the encapsulation efficiency was determinedby taking the ratio of the final and initial siRNA-to-lipid ratio andmultiplying the result by 100 to obtain a percentage.

In Vivo Screening of Cationic Lipids for FVII Activity

FVII activity was evaluated in FVII siRNA-treated animals at 24 hoursafter intravenous (bolus) injection in C57BL/6 mice or 48 hours afterintravenous (bolus) injection in SD rats. Six to 8 week old, femaleC57BI/6 mice were obtained from Charles River Laboratories andacclimated for one week prior to use in studies. Animals were held in apathogen-free environment and all procedures involving animals wereperformed in accordance with the guidelines established by the CanadianCouncil on Animal Care.

LN-siRNA systems containing Factor VII siRNA were diluted to theappropriate concentrations in sterile phosphate buffered salineimmediately prior to use, and the formulations were administeredintravenously via the lateral tail vein in a total volume of 10 ml/kg.After 24 h, animals were anaesthetised with Ketamine/Xylazine and bloodwas collected by cardiac puncture and processed to serum (MicrotainerSerum Separator Tubes; Becton Dickinson, Franklin Lakes, N.J., USA).Serum was tested immediately or stored at −70° C. for later analysis forserum Factor VII levels.

FVII was measured using a commercially available kit (Biophen FVII Kit™;Aniara Corp., Mason, Ohio), following the manufacturer's instructions ata microplate scale. FVII reduction was determined against untreatedcontrol animals, and the results were expressed as % Residual FVII. Fivedose levels (0.1, 0.3, 0.5, 1.0, and 3.0 mg/kg) were typically used.

Pharmacokinetic and Liver Analysis

A fluorescently labeled siRNA (Cy-3 labeled luciferase siRNA, AlnylamPharmaceuticals) was used to measure the siRNA content in plasma andliver after iv administration of LN-siRNA systems. The measurements weredone by first extracting the Cy3-siRNA from the protein-containingbiological matrix and then analyzing the amount of Cy-3 label in theextract by fluorescence. Two extraction methods were used, achloroform/methanol mixture for the plasma samples and a commercialphenol/chloroform mixture (Trizol® reagent) with the tissue samples.

For plasma, blood was collected in EDTA-containing Vacutainer tubes andcentrifuged at 1000×g for 10 min at 4-8° C. to isolate the plasma. Theplasma was transferred to an eppendorf tube and either assayedimmediately or stored in a −30° C. freezer. An aliquot of the plasma(100 μL maximum) was diluted to 500 μL with PBS (145 mM NaCl, 10 mMphosphate, pH 7.5), methanol (1.05 mL and chloroform (0.5 mL) was added,and the sample vortexed to obtain a clear, single phase solution. Water(0.5 mL) and chloroform (0.5 mL) was then added and the resultingemulsion sustained by mixing periodically for a minimum of 3 minutes.The mixture was centrifuged at 3000 rpm for 20 minutes and the topaqueous phase containing the Cy-3-label transferred to a new test tube.The fluorescence of the solution was measured using an SLM Fluorimeterat an excitation wavelength of 550 nm (2 nm bandwidth) and emissionwavelength of 600 nm (16 nm bandwidth). A standard curve was generatedby spiking aliquots of plasma from untreated animals with the Cy-3-siRNAcontaining formulation (0 to 15 μg/mL), and the sample processed asindicated above.

For liver, sections (400-500 mg) of tissue from saline-perfused animalswas accurately weighed and homogenized in 1 mL of Trizol using Fastpreptubes. An aliquot of the homogenate (typically equivalent to 50 mg oftissue) was transferred to an eppendorf tube and additional Trizol addedto 1 mL final. Chloroform (0.2 mL) was added, and the solution was mixedand incubated for 2-3 min before being centrifuged for 15 min at12,000×g. An aliquot (0.5 mL) of the top Cy-3-containing aqueous phasewas diluted with 0.5 mL of PBS and the fluorescence of the samplemeasured as described above.

Measurement of FVII Protein in Serum

Serum Factor VII levels were determined using the colorimetric BiophenVII assay kit (Anaira, USA). Briefly, serially diluted pooled controlserum (200%-3.125%) and appropriately diluted plasma samples fromtreated animals were analyzed using the Biophen VII kit according tomanufacturer's instructions in 96-well, flat bottom, non-bindingpolystyrene assay plates (Corning, Corning, N.Y.) and absorbance at 405nm was measured. A calibration curve was generated using the serialdiluted control serum and used to determine levels of Factor VII inserum from treated animals.

Determination of Tolerability

The tolerability of empty DLin-K-C2-DMA lipid particles(DLin-K-C2-DMA/DSPC/Chol/PEG-C-DOMG (40/10/40/10)) was evaluated bymonitoring weight change, cageside observations, clinical chemistry and,in some instances, hematology in femal Sprague Dawley rats and femaleC57BL/6 mice. Animal weights were recorded prior to treatment and at 24hours after intravenous treatment with various dosages. Data wasrecorded as % Change in Body Weight. In addition to body weightmeasurements, clinical chemistry panel, including liver functionmarkers, was obtained at each dose level at 24 hours post-injectionusing an aliquot of the serum collected for FVII analysis. Samples weresent to the Central Laboratory for Veterinarians (Langley, B C) foranalysis. In some instances, additional animals were included in thetreatment group to allow collection of whole blood for hematologyanalysis.

In Situ Determination of pKa Using TNS

In situ pKa measurements were made using the pH sensitive fluorescentprobe TNS, using a modification of the approach previously published inBailey, A. L. and Cullis, P. R., Biochemistry 33:12573-12580 (1994).

Results

Initial studies were performed in eight to 10 week old, female C57BL/6mice in two stages. In the first stage, the activity associated with thebenchmark lipid DLinDMA was compared to the activity associated withmodified forms of DLinDMA. This stage resulted in the identification ofDLin-K-DMA as having increased activity as compared to DLinDMA.Therefore, in the second stage, the activities of modified forms ofDLinDMA were compared to the activity of DLin-K-DMA. Dose responsecurves were used to estimate an ED₅₀ for each formulation, which isdefined as the siRNA dose required to reduce the concentration of serumFVII protein by 50%, and is ˜1.0 mg/kg for the DLinDMA benchmarkformulation. The ED₅₀ for formulations with poor activity is expressedas a range, e.g. 12-25 mg/kg, indicating that a 50% reduction in serumFVII protein levels occurs between these siRNA doses. If a formulationshowed good activity (ED₅₀<2 mg/kg), then the dose response was repeatedover a narrower dose range and head-to-head with DLinDMA for greatercomparative accuracy.

Screen 1a: Headgroup Modifications to DLinDMA and In Vivo FVII Activity

For the purposes of this study, DLinDMA was divided into three keystructural domains that were modified separately, including theheadgroup, the linker, and the hydrocarbon chains. Thedimethylaminopropane headgroup is hydrophilic and contains a tertiaryamine function with an apparent pKa (in situ) of pH 6.4. Consequently,DLinDMA is almost completely charged at pH 4, the pH at which theLN-siRNA systems are formed through electrostatic interaction withsiRNA. Whereas, at pH 7.4, ˜5-10% of DLinDMA molecules are charged;therefore, the cationic charge density at the surface of thesenanoparticles in the circulation is relatively low. In contrast, atendosomal pH's (˜pH 5), the surface charge density is increasedsignificantly, which is expected to promote the formation of ion pairswith anionic phospholipids and disrupt the endosomal membrane (Hafez, I.M. and Cullis, P. R., Adv. Drug Deliv. Rev. 4:139-148 (2001) and Xu, Y.and Szoka, F. C., Biochemistry 35:5616-5623 (1996)).

The headgroup modifications made to DLinDMA are shown in Table 3, andthe first three were designed to alter the nature of the positivecharge. DLinTMA contains a quaternary amino group and is permanentlycharged, and showed reduced activity with an estimated ED₅₀ between 2-5mg/kg. A similar decrease in activity was observed when thedimethylamine function was replaced by a piperazine moiety (DLinMPZ,ED₅₀ 2-5 mg/kg), and reduced more significantly when substituted by amorpholino group (DLinMA, ED₅₀ 12-25 mg/kg).

One rationale for making the remaining two modifications shown in Table3 was to compare the activities of two lipids with similar headgroupstructures but with different rates of metabolism in vivo. Lipiddegradation was not measured in vivo for any of the lipids screenedhere, but it is expected that an ethoxy group (DLin-EG-DMA) would bemore resistant to enzymatic cleavage than an ester group (DLinDAC)(Martin, B. et al., Curr. Pharm. Des 11:375-394 (2005)); none the less,both lipids exhibited similar activity.

TABLE 3 Headgroup modifications to DLinDMA Abbreviated ED₅₀ NameChemical Name (mg/kg) Headgroup Modification DLinDMA (Benchmark)1,2-Dilinoleyloxy-3- dimethylaminopropane ~1

DLinTMA.Cl 1,2-Dilinoleyloxy-3- trimethylaminopropane chloride 2-5

DLinMPZ 1,2-Dilinoleyloxy-3-(N- methylpiperazino) propane 2-5

DLinMA 1,2-Dilinoleyloxy-3- morpholinopropane 12-25

DLin-EG-DMA 1,2-Dilinoleyloxy-3-(2- N,N-dimethylamino) ethoxypropane5-12

DLinDAC 1,2-Dilinoleyloxy-3- (dimethyalmino) acetoxypropane 5-12

Screen 1b: Linker Modifications to DLinDMA and In Vivo FVII Activity

DLinDMA has two unsaturated hydrocarbon chains joined to thedimethylaminopropane headgroup through two ethoxy linkages. In a bilayerstructure, the linker region resides at the membrane interface, an areaof transition between the hydrophobic membrane core and hydrophilicheadgroup surface. The approach to linker modification of DLinDMA was tointroduce linker groups expected to exhibit different rates of chemicalor enzymatic stability and spanning a range of hydrophilicity. Theethoxy moiety is considered to be more resistant to degradation thanmost other types of chemical bonds in vivo, which may be why theselipids have been found to be less well tolerated than cationic lipidswith ester linkages for example (Martin, B. et al., Curr. Pharm. Des11:375-394 (2005)). A variety of these rationally designed lipids weremade, characterized, and tested, including those shown in Table 4.

TABLE 4 Linker modifications to DLinDMA Abbreviated ED₅₀ Name ChemicalName (mg/kg) Structure DLinDMA (Benchmark) 1,2-Dilinoleyloxy-3-dimethylaminopropane ~1

DLinDAP 1,2-Dilinoleoyl-3- dimethylaminopropane 40-50

DLin-2-DMAP 1-Linoleoyl-2-linoeyloxy- 3-dimethylaminopropane  5-12

DLin-C-DAP 1,2- Dilinoleylcarbamoyloxy- 3-dimethylaminopropane 12-25

DLin-S-DMA 1,2-Dilinoleylthio-3- dimethylaminopropane 12-25

DLin-K-DMA 2,2-Dilinoleyl-4- dimethylaminomethyl- [1,3]-dioxolane ~0.4

The first modification listed in Table 4 is DLinDAP, in which estersreplace the ethoxy linkers of DLinDMA. Remarkably, nucleic acid lipidparticles comprising Factor VII siRNA and containing DLinDAP showedsignificantly reduced in vivo activity as compared to those containingDLinDMA (ED₅₀ 12-25 mg/kg), despite its very similar structure toDLinDMA. Further, nucleic acid-lipid particles based on DLin-2-DMAP, alipid with one ethoxy linkage and one ester linkage, yielded activityintermediate between DLinDAP- and DLinDMA-based nucleic acid-lipidparticles. Nucleic acid-lipids particles based on lipids containingcarbamate (DLin-C-DAP) or thioether (DLin-S-DMA) linkages also resultedin dramatically reduced in vivo activity.

The final modification was to insert a ketal ring linker, whichintroduced interesting structural changes to the lipid molecule. First,the ketal is known to be more acid labile than ethoxy linkers (Martin,B. et al., Curr. Pharm. Des 11:375-394 (2005)), which may decrease itshalf-life in the endocytic pathway. Second, the hydrocarbon chains nowbond to the linker group through a single carbon. Interestingly, theintroduction of a ketal ring linker into DLinDMA resulted in nucleicacid-lipid particles that were ˜2.5-fold more potent in reducing serumFVII protein levels relative to the DLinDMA benchmark, with an ED₅₀(i.e., dose to achieve 50% gene silencing) of ˜0.4 mg/kg versus 1 mg/kg,respectively (FIG. 2).

Screen 1c: Reduce Unsaturation of DLinDMA Hydrocarbon Chains,Miscellaneous Modifications and In Vivo FVII Activity

A variety of other cationic lipids containing modifications as comparedto DLinDMA were tested in the Factor VII knockdown system. For example,the propensity of lipid molecules to adopt inverted non-bilayer phasesis known to increase with increasing hydrocarbon chain unsaturation(Cullis, P. R., et al., Chem. Phys. Lipids 40:127-144 (1986)). Given thehypothesis that formation of these non-bilayer phases is responsible forendosome disruption and release of siRNA into the cytoplasm and theobservation that SNALP activity in vitro also increases with increasingunsaturation (Heyes, J. et al., J. Control Release 107:276-287 (2005)),it was of interest to see what happened to LN-siRNA potency in vivo whenDLinDMA, containing two C18:2 chains was replaced by DODMA, with twoC18:1 chains. These cationic lipids and the results of these experimentsare shown in Table 5.

TABLE 5 Miscellaneous modifications to headgroup, linker and hydrocarbonchains Abbreviated ED₅₀ Name (mg/kg) Modification DLinDMA (Benchmark) ~1

DODMA 2-5

DODAP >25

DO-C-DAP >10

DMDAP >10

DLinTAP.Cl >25

DOTAP.Cl >25

DLinAP 5-12

As shown in Table 5, DODMA was 2 to 5-fold less active than the moreunsaturated DLinDMA, and substituting ether linkages for esters (DODAP)decreased activity more than 25-fold.

Carbamate linked C18:1 chains (DO-C-DAP) were also an inactivecombination at 10 mg/kg, the maximum dose tested. DMDAP was synthesizedto determine if the shorter C14:1 hydrocarbon chains might enable theester-linked lipid through enhanced lipid mixing with the targetendosomal membrane (Mui, B. et al., Biochim. Biophys. Acta 1467:281-292(2000)); however, no activity was observed for DMDAP-LN-siRNA in theFVII model up to a maximum dose of 10 mg/kg. The permanently charged,ester linked lipids DLinTAP and DOTAP were of interest, because thelatter lipid is one of the most commonly used cationic lipids fortransfection. However, in the LN-siRNA model, neither of theseester-linked lipids showed any signs of activity, (ED₅₀>25 mg/kg). Thelast lipid represented a radical structural change to DLinDMA, in whichthe dimethylpropane headgroup was reversed and the hydrocarbon chainsbond directly to the amino nitrogen, leaving a dihydroxy headgroup;however, DLinAP showed poor activity with an ED₅₀ in the 5-12 mg/kgrange.

In summary, the incremental modifications to DLinDMA successfullyidentified DLin-K-DMA as a cationic lipid that is significantly morepotent than DLinDMA when tested head-to-head in the same in vivo modeland LN-siRNA formulation.

Screen 2a: Headgroup Modifications to DLin-K-DMA and In Vivo FVIIActivity

Given the importance of positive charge in the mechanism of actionhypothesis guiding the lipid design, the effects of structural changesin the amine-based headgroup were investigated in the context ofDLin-K-DMA as the new benchmark lipid. A series of headgroupmodifications were made, characterized, and tested to explore the effectof size, acid dissociation constant, and number of ionizable groups(Table 6).

TABLE 6 Headgroup modifications to DLin-K-DMA Chemical ED₅₀ AbbreviatedName Name (mg/kg) Modification DLin-K-DMA (Benchmark) 2,2-Dilinoleyl-4-dimethylamino methyl-[1,3]- dioxolane ~0.4

DLin-K-MPZ 2,2-Dilinoleyl-4- N-methyl piperazino-[1,3]- dioxolane ~1.5

DLin-K-MA 2,2-Dilinoleyl-4- N-morpholino- [1,3]-dioxolane >15

DLin-K-TMA.Cl 2,2-Dilinoleyl-4- trimethylamino- [1,3]-dioxolane Chloride>5^(a)

DLin-K²-DMA 2,2-Dilinoleyl- 4,5-bis (dimethylamino methyl)-[1,3]-dioxolane ~0.4

DLin-K-C2-DMA 2,2-Dilinoleyl- 4-(2-dimethyl aminoethyl)-[1,3]- dioxolane~0.1

DLin-K-C3-DMA 2,2-Dilinoleyl- 4-(3-dimethyl aminopropyl)-[1,3]-dioxolane ~0.6

DLin-K-C4-DMA 2,2-Dilinoleyl- 4-(4-dimethyl aminobutyl)- [1,3]-dioxolane>3

^(a)No activity observed at 5 mg/kg and lethal at next dose of 15 mg/kg

DLin-K-DMA contains a chiral carbon at position 4 of the ketal ringstructure. Therefore, the two optically pure (+) and (−) enantiomerswere synthesized and their activities compared to that of the racemicmixture. All three formulations exhibited indistinguishable doseresponses, each with an ED₅₀˜0.3 mg/kg.

The first three modifications listed in Table 6 were also applied toDLinDMA in screen 1, the introduction of piperazino (DLin-K-MPZ) andmorpholino (DLin-K-MA) amino moieties to modify the characteristics ofthe ionizable positive charge, and also converting the tertiarydimethylamine into the permanently charged quaternary amine ofDLin-K-TMA. Although all these modifications significantly decreasedactivity, it is interesting to note that DLin-K-MPZ, with an ED₅₀˜1.5,was almost as active as DLinDMA but approximately 5-fold less activethan DLin-K-DMA, and the same modification to DLinDMA reduced itsactivity by a similar factor. Furthermore, the morpholino amine functionmade DLin-K-MA inactive at the maximum dose tested (15 mg/kg) similar toDLinMA, which has an ED₅₀ of 12-25 mg/kg. Another observation of note isthat DLin-K-TMA (permanent positive charge) is toxic. No activity wasobserved at 5 mg/kg, but animals experienced significant weight loss(data not shown) and did not survive the next dose at 15 mg/kg.

The modification abbreviated as DLin-K²-DMA denotes the presence of twodimethylamine moieties. Within error, this lipid had the same activityas DLin-K-DMA, despite the larger headgroup.

As an additional parameter, the distance between the dimethylamino groupand the dioxolane linker was varied by introducing additional methylenegroups. The remaining three lipids were closely related in structure,and were synthesized to determine what effect distancing the positivecharge from the dioxolane ring had on activity. This parameter canaffect both the pK_(a) of the amine headgroup as well as the distanceand flexibility of the charge presentation relative to the lipid bilayerinterface. Inserting a single additional methylene group into theheadgroup (DLin-K-C2-DMA) produced a dramatic increase in potencyrelative to DLin-K-DMA. The ED₅₀ for this lipid was ˜0.1 mg/kg, makingit 4-fold more potent than DLin-K-DMA and 10-fold more potent than theDLinDMA benchmark when compared head-to-head in the FVII model (FIG.2A). Additional methylene groups decreased activity with a significantreduction occurring between DLin-K-C3-DMA (ED₅₀˜0.6 mg/kg) andDLin-K-C4-DMA (ED₅₀>3 mg/kg) (FIG. 2B).

Screen 2b: Modifications to the Headgroup, Linker and Hydrocarbon Chainsof DLin-K-DMA and In Vivo FVII Activity

A number of additional structural modifications made to DLin-K-DMA arepresented in Table 7. The first three in the series confirmed theimportance of hydrocarbon chain unsaturation for in vivo activity. Aprogressive decrease in ED₅₀'s from ˜0.3, to ˜1.0 and ˜8.0 mg/kg wasobserved going from DLin-K-DMA (C18:2) to DO-K-DMA (C18:1) and DS-K-DMA(C18:0), respectively. The next modification (DLin-K6-DMA) demonstratedthat the 5-membered ketal ring of DLin-K-DMA could be substituted by a6-membered dioxolane ring structure without loss of activity. The finallipid shown in Table 7 represented a more radical modification.DLin-M-DMA does not have a ketal ring linker, but the hydrocarbon chainsstill bond directly with a single carbon. Interestingly, this lipidremained relatively active with an ED₅₀˜0.7 mg/kg.

TABLE 7 Miscellaneous modifications to headgroup, linker and hydrocarbonchains of DLin-K-DMA Abbreviated ED₅₀ Name (mg/kg) ModificationDLin-K-DMA (Benchmark) ~1

DO-K-DMA ~1.0

DS-K-DMA ~8.0

DLin-K6-DMA ~0.3

DLin-M-DMA ~0.7

Comparison of the In Vivo Activity of Nucleic Acid-Lipid Formulations inMice and Rats

The ability of various nucleic acid-lipid formulations comprisingdifferent cationic lipids was further explored in mice and rats. Each ofthe tested nucleic acid-lipid formulations was prepared as describedabove using PEG-C-DOMG as the PEG-lipid. The formulations initiallytested (which included either DLin-K-DMA, DLin-K-MPZ, DLin-K-C2-DMA, orDLin-K-C4-DMA) reduced residual FVII levels in both mice (FIG. 3) andrats (FIG. 4). However, the DLin-K-C2-DMA formulation showed aremarkably enhanced ability to reduce FVII levels in both mice and rats.The activity of the DLin-K-C2-DMA formulation was approximately 2-3-foldgreater than the DLin-K-DMA formulation in mice, and approximately10-20-fold greater than the DLin-K-DMA formulation in rats. A comparisonof FVII reduction in mice and rats using the DLin-K-DMA formulation orthe DLin-K-C2-DMA formulation is shown in FIG. 5. Formulations havingDLin-K-C4-DMA or DLin-K-MPZ (MPZ) as the cationic lipid showed similaractivity to each other and to the DLinDMA formulation.

The liposomal formulation having DLin-K6-DMA as the cationic lipid wasalso tested in comparison to DLin-K-C2-DMA and DLin-K-DMA. TheDLin-K6-DMA formulation reduced FVII levels in mice similarly toDLin-K-DMA, as shown in FIG. 6.

Pharmacokinetics and Liver Accumulation of Cationic LN-siRNAFormulations

The correlation between levels of siRNA delivered to the liver and FVIIreduction was determined by encapsulating Cy-3 labeled siRNA in theselection of LN-siRNA systems covering a spectrum of in vivo activitiesshown in Table 8. Cy-3 fluorescence was measured in plasma and livertissue 0.5 and 3.0 h post injection. The plasma data indicated a widevariety of clearance rates at the early time point, but for the majorityof the formulations, 20-50% of the injected siRNA dose was recovered inthe liver within 0.5 h, whether they were highly active or exhibitedpoor activity. The most active formulations, DLinDMA and DLin-K-DMA,showed relatively high levels of siRNA in the liver at 0.5 h, 50% and32% respectively. All formulations showed a decrease in liver Cy-3levels after 3 h, which presumably reflected metabolism. This studysuggests that gross delivery to the liver alone does not explaindifferences in activity.

TABLE 8 Plasma and liver concentrations of Cy-3 siRNA for a selection ofactive and inactive cationic lipid-containing LN-siRNA systems 0.5 h (%injected dose) 3.0 h (% injected dose) ED₅₀ Lipid Plasma Liver PlasmaLiver (mg/kg) DLin-K-DMA 1.1 32.0 0.4 4.0 0.3 DLinDMA 15.3 50.0 0.7 17.01.0 DLinMPZ 20.3 52.0 0.4 37.5 2-5 DLinAP 86.2 11.5 23.1 5.0  5-12DLin-2-DMAP 17.5 20.5 8.8 2.5  5-12 DLinDAC 27.1 29.0 0.3 6.5 12-25DLinDAP 46.6 20.5 3.3 16.5 12-25 DLin-C-DAP 69.4 28.5 19.0 13.5 12-25DLin-S-DMA 10.7 2.5 5.4 0 12-25 DLinMA 20.2 10.5 0.4 4.5 12-25

Tolerability of DLin-K-C2-DMA-Containing LN-siRNA Systems

Rats administered the liposomal formulation containing DLin-K-C2-DMAshowed a dose-dependent loss of weight. Rats administered 91 mg/kgappeared normal and had normal livers. Rats administered 182 mg/kgshowed slower movement and a scruffy coat. Their livers were slightlypale, and one of three livers showed some slight mottling. Of the ratsadministered 364 mg/kg, one died, and they showed hunched, slowermovement, quinting eyes, scruffy coats, piloerection, red/orange urine,with pale and some mottling livers. Rats showed significant increases inALT/AST, as low as 182 mg/kg lipid.

Histopathology results for livers obtained from rats treated with 91mg/kg (“5” mg/kg) were normal. The livers of rats treated with 182 mg/kg(“10” mg/kg) showed mild to moderate hepatocellular necrosis,centrilobular, and hepatocellular vacuolization. One of the livers ofthe surviving rats treated with 364 mg/kg (“20” mg/kg) showed moderatehepatocellular necrosis, centrilobular, and the other showed diffuse,mild to moderate hepatocellular necrosis (not concentrated incentrilobular region) with mild inflammation.

Mice treated with the liposomal formulation of DLin-K-C2-DMA also showeda dose-dependent loss of weight, although no mice died. Mice also showeda greater than 10-fold increase in ALT-AST at approximately 1100 mg/kglipid. However, the mice showed no obvious clinical signs, except atgreater than 1300 mg/kg, where the mouse exhibited hunched, slowermovement and a scruffy coat.

The introduction of a ketal linker did not appear to impart anysignificant toxicity issues in mice and, in fact, the LN systemscontaining DLin-K-DMA and encapsulating FVII siRNA were extraordinarilywell tolerated in mice. The data presented in Table 9 are from a studydesigned to determine appropriate dosing ranges, and extreme, singledoses of lipid and siRNA were achieved. The toxicity criteria measuredwere % change in body weight and serum levels of the liver enzymemarkers, ALT and AST.

TABLE 9 Key tolerability parameters for DLin-K-DMA-containing LN-siRNAsystems in mice at extreme doses siRNA Total Lipid % Change ALT AST(mg/kg) (mg/kg) Body Weight (IU/L) (IU/L) 0-10 0-164 0 45 90 46 750 −5.0174 340 61 1000 −4.5 448 816 76 1250 −5.5 1771 4723 92 1500 −6.0 47232094Compared to saline controls, no changes in blood chemistry or bodyweight were observed up to an siRNA dose of 10 mg/kg, which for thisformulation was >30-fold greater than the ED₅₀ dose.

A massive siRNA dose of 46 mg/kg (150-fold more than the ED₅₀) wasadministered before significant toxicity signs were measured. This siRNAdose translated to a total lipid dose of 750 mg/kg at the siRNA-to-lipidratio of 0.06 (wt/wt). Even at these levels, the increases in serum ALTand AST were relatively modest (<10-fold normal), and it is not untilsiRNA doses exceeded 61 mg/kg that severe (>10-fold) increases areobserved. The maximum dose tested was 92 mg siRNA/kg, equivalent to 1500mg total lipid/kg. Animals lost 6% body weight but no deaths occurred atany of the doses tested.

Characterization of Nucleic Acid-Lipid Particles

Characteristics of selected nucleic acid-lipid formulations aresummarized in Table 10, wherein C2 indicates that the cationic lipid isDLin-K-C2-DMA; C4 indicates that the cationic lipid is DLin-K-C4-DMA;and MPZ indicates that the cationic lipid is DLin-K-MPZ. Each of theformulations described below contained PEG-C-DOMG as the PEG-lipid.

TABLE 10 Characteristics of Formulations Comprising Various Amino LipidsFinal Lipid Ratio (mol %) Particle Final Formu- PEG-C- Size D/L Ratio %Free lation Cat DSPC Chol DOMG (nm) (wt/wt)* siRNA C2 37.4 11.5 41.3 9.872 ± 23 0.037 7.8 C4 37.9  9.6 42.8 9.7 64 ± 15 0.058 1.2 MPZ ND ND NDND 71 ± 21 0.056** 5.1 *Based on encapsulated material; **estimated fromDSPC, Chol, PEG-C-DOMG results

Apparent pKa's of Key Cationic Lipids Measured In Situ in LN-siRNAFormulations

Two important parameters underlying the lipid design are the pK_(a) ofthe ionizable cationic lipid and the abilities of these lipids, whenprotonated, to induce a non-bilayer (hexagonal H_(II)) phase structurewhen mixed with anionic lipids. The pK_(a) of the ionizable cationiclipid determines the surface charge on the LNP under different pHconditions. The charge state at physiologic pH (e.g., in circulation)can influence plasma protein adsorption, blood clearance and tissuedistribution behavior (Semple, S. C., et al., Adv. Drug Deliv Rev32:3-17 (1998)), while the charge state at acidic pH (e.g., inendosomes) can influence the ability of the LNP to combine withendogenous anionic lipids to form endosomolytic non-bilayer structures(Hafez, I. M., et al., Gene Ther 8:1188-1196 (2001)). Consequently, theability of these lipids to induce H_(II) phase structure in mixtureswith anionic lipids is a measure of their bilayer destabilizing capacityand relative endosomolytic potential.

The fluorescent probe 2-(p-toluidino)-6-napthalene sulfonic acid (TNS),which exhibits increased fluorescence in a hydrophobic environment, canbe used to assess surface charge on lipid bilayers. Titrations ofsurface charge as a function of pH can then be used to determine theapparent pK_(a) (hereafter referred to as pK_(a)) of constituent lipids(Cullis, P. R., et al., Chem Phys Lipids 40:127-144 (1986)). Using thisapproach, the pK_(a) values for nucleic acid-lipid particles containingvarious cationic lipids were determined and are summarized in Table 11.The relative ability of the protonated form of certain ionizablecationic lipids to induce H_(II) phase structure in anionic lipids wasascertained by measuring the bilayer-to-hexagonal H_(II) transitiontemperature (T_(BH)) in equimolar mixtures withdistearoylphosphatidylserine (DSPS) at pH 4.8, using ³¹P NMR (Cullis, P.R. and de Kruijff, B., Biochim Biophys Acta 513:31-42 (1978)) anddifferential scanning calorimetric (DSC) analyses (Expand, R. M. et al.,Biochemistry 28:9398-9402 (1989)). Both techniques gave similar results.

The data presented in Table 11 indicate that the highly active lipidDLin-K-C2-DMA has pK_(a) and T_(BH) values that are theoreticallyfavorable for use in siRNA delivery systems. The pK_(a) of 6.4 indicatesthat LNPs based on DLin-KC2-DMA have limited surface charge incirculation, but will become positively charged in endosomes. Further,the T_(BH) for DLin-K-C2-DMA is 7° C. lower than that for DLinDMA,suggesting that this lipid has improved bilayer destabilizing capacity.However, the data also demonstrate that pK_(a) and T_(BH) do not fullyaccount for the in vivo activity of lipids used in LNPs. For example,DLin-K-C3-DMA and DLin-K-C4-DMA have identical pK_(a) and T_(BH) values,yet DLin-KC4-DMA is more than 5-fold less active in vivo. Moreover,DLin-K-C2-DMA and DLin-K-C4-DMA, which have very similar pK_(a) andT_(BH) values, exhibit a >30-fold difference in in vivo activity. Thus,while the biophysical parameters of pK_(a) and T_(BH) are useful forguiding lipid design, the results presented in Table 11 support thestrategy of testing variants of lead lipids, even ones with very similarpK_(a) and T_(BH) values.

TABLE 11 pKa's of key cationic lipids measured in situ in preformedvesicles using TNS fluorescence titrations H_(II) transition temperatureFVII ED₅₀ Cationic Lipid Apparent pKa (° C.) (mg/kg) DLin-K-C3-DMA 6.818 ~0.6 DLin-K-C4-DMA 6.8 18 >3.0 DLinDMA 6.4 27 ~1.0 DLin-K-C2-DMA 6.420 ~0.1 DLin-K6-DMA 6.2 n.d. ~0.3 DLin-K-MPZ 6.2 n.d. ~1.5 DLinDAP 5.726 >25 DLin-K-DMA (racemic 5.6 n.d. ~0.3 mixture) DLin-K-MA 5.6 n.d. >15DLin-K-DMA 5.6 19 ~0.4

As shown above, the potency of LN-siRNA systems containing 40 mole %DLin-K-C2-DMA was such that as little as ˜100 picomoles of encapsulatedsiRNA administered as a single i.v. bolus to a mouse was sufficient toknockdown serum concentrations of FVII protein by 50% within 24 h ofinjection.

In this study, the ratios of lipid components and siRNA-to-lipid werekept constant for all formulations, so that any differences in surfacecharge could be attributed to cationic lipid pKa. The siRNA-to-lipidratio used in the activity screen was 0.06 wt/wt, which means thatpositive charge was in excess of negative charge. Charge neutralizationfor formulations containing 40 mole % monobasic cationic lipid occurs ata ratio of approximately 0.17 wt/wt. Consequently, assuming one cationiclipid forms an ion pair with each negative charge on the siRNA backbone,then approximately 35% of the total cationic lipid was associated withsiRNA inside the nanoparticle and, therefore, could not contribute tosurface charge. Interestingly, increasing the siRNA-to-lipid ratio above˜0.08 (wt/wt) in a 40/10/40/10 formulation decreased the potency ofLN-siRNA systems (data not shown). A similar response has been reportedfor siRNA delivered in vivo using lipidoid nanoparticles (A. Akinc, M.et al., Mol. Ther. (2009) and may reflect the importance of freecationic lipid (lipid not associated with siRNA) and/or the totalcationic lipid dose injected.

One of the most striking observations was the dependence of activity onthe extent of hydrocarbon chain unsaturation. For both DLinDMA andDLin-K-DMA, there was a significant decrease in potency for each drop inthe number of double bonds. Without wishing to be bound by theory, it isproposed that (active) synthetic cationic lipid inserted into theendosomal membrane and endogenous anionic phospholipids (such asphosphatidylserine) form ion pairs (Hafez, I. M., et al., Gene Ther.8:1188-1196 (2001) and Xu, Y. and Szoka, F. C., Biochemistry35:5616-5623 (1996)). The resulting charge neutralization effectivelyreduces the cross-sectional area of the combined headgroups, whichcorresponds to a substantial decrease in their intrinsic radius ofcurvature. Applying the molecular shape arguments employed to describelipid polymorphism, this means the cationic and anionic lipids go from acylindrical shape they adopt in isolation to a cone shape formed by theneutral ion pair. Cylindrical shaped lipids are compatible with bilayerstructure, whereas cone shaped lipids are not Cullis, P. R. et al.,Chem. Phys. Lipids 40:127-144 (1986) and Hafez, I. M. and Cullis, P. R.,Adv. Drug Deliv. Rev. 47:139-148 (2001)), they prefer to adopt invertedlipid phases, such as the hexagonal H_(II) phase, that disrupt bilayerstructure. As a consequence, the endosome membrane is lysed, enablingsiRNA to access the cytoplasm where it can engage RISC and cleave FVIImRNA.

The results described herein are consistent with the shape conceptintroduced above, because adding cis double bonds to a given chainlength increased the cross-sectional area swept out by the terminalmethyl groups, thus promoting cone-like geometry. The propensity toadopt non-bilayer structures when paired with anionic phospholipids isalso a plausible rationale for why the ketal-containing lipid family isso active. Both hydrocarbon chains bond through a single carbon into theketal ring linker, the tetrahedral bond angle will tend to splay thechains apart favoring a cone shape.

Example 17 Efficacy and Tolerability of the DLin-K-C2-DMA SNALPFormulation

The efficacy and tolerability of nucleic acid lipid particles comprisingDLin-K-C2-DMA was further validated in the context of nucleic acid-lipidparticles formulated for delivery of siRNA in vivo, termed KC2-SNALP.These particles comprise a different ratio of lipids than thePFV-prepared nucleic acid-lipid particles described in Example 16.

siRNA were encapsulated in SNALP using a controlled step-wise dilutionmethod process described by Jeffs et al. (Pharm Res 22:362-372 (2005))The lipid constituents of KC2-SNALP were DLin-KC2-DMA (cationic lipid),dipalmitoylphosphatidylcholine (DPPC; Avanti Polar Lipids, Alabaster,Ala.), synthetic cholesterol (Sigma, St. Louis, Mo.) and PEG-C-DMA usedat a molar ratio of 57.1:7.1:34.3:1.4, respectively. Upon formation ofthe loaded particles, SNALP were dialyzed against PBS and filtersterilized through a 0.2 μm filter before use. Mean particle sizes were75-85 nm and 90-95% of the siRNA was encapsulated within the lipidparticles. The final lipid-to-siRNA ratio in formulations used for invivo testing was approximately 6.5:1 (wt:wt).

The KC2-SNALP formulation showed a marked improvement in potency in themouse FVII model as compared to the DLin-K-C2-DMA formulation describedin Example 16. The measured ED₅₀ decreased from ˜0.1 mg/kg for theDLin-K-C2-DMA nucleic acid-lipid formulation described in Example 16 to˜0.02 mg/kg for the KC2-SNALP formulation (FIG. 8A). KC2-SNALP was alsofound to exhibit similar potency in rats (data not shown).

In addition to efficacy, tolerability is another critical attribute of asuitable nucleic acid-lipid particle delivery system for human use, sothe single-dose tolerability of KC2-SNALP was studied in rats. Dosesnear the efficacious dose level were found to be very well tolerated(data not shown); therefore, single-dose escalation studies wereconducted starting at doses ˜50-fold higher (1 mg/kg) than the observedED₅₀ of the formulation. To understand formulation toxicity in theabsence of any toxicity or pharmacologic effects resulting from targetsilencing, these experiments were conducted using the non-targetingcontrol siRNA sequence directed against luciferase described in Example16. Clinical signs were observed daily, and body weights, serumchemistry, and hematology parameters were measured at 72 hourspost-dose. As shown in Table 12, KC2-SNALP was very well tolerated atthe high dose levels examined (relative to the observed ED₅₀ dose) withno dose-dependent, clinically significant changes in key serum chemistryor hematology parameters.

TABLE 12 Clinical chemistry and hematology parameters in rats siRNATotal dose ALT AST Bilirubin BUN RBC Hemoglobin WBC PLT Vehicle (mg/kg)(U/L) (U/L) (mg/dL) (mg/dL) (×10⁶/μL) (g/dL) (×10³/μL) (×10³/μL) PBS 56± 16 109 ± 31 2 ± 0 4.8 ± 0.8 5.5 ± 0.3 11.3 ± 0.4 11 ± 3 1166 ± 177KC2-SNALP 1 58 ± 22 100 ± 14 2 ± 0 4.4 ± 0.6 5.6 ± 0.2 11.6 ± 0.6 13 ± 21000 ± 272 KC2-SNALP 2 73 ± 9   81 ± 10 2.2 ± 0.4 4.3 ± 0.6 5.9 ± 0.311.6 ± 0.3 13 ± 4 1271 ± 269 KC2-SNALP 3 87 ± 19 100 ± 30 2 ± 0 5.0 ±0.8 6.0 ± 0.2 11.9 ± 0.4 15 ± 2  958 ± 241

Example 18

In Vivo Efficacy and Tolerability of KC2-SNALP in Primates

Given the promising activity and safety profile observed in rodents inthe studies described in Example 17, studies were performed in non-humanprimates to investigate the translation of DLin-KC2-DMA activity inhigher species. For these studies, transthyretin (TTR), a hepatic geneof high therapeutic interest, was targeted.

Cynomolgus monkeys were treated with a single 15 minute intravenousinfusion of KC2-SNALP-formulated siTTR at siRNA doses of 0.03, 0.1, 0.3and 1 mg/kg. Control animals received a single 15 minute intravenousinfusion of PBS or KC2-SNALP-formulated ApoB siRNA at a dose of 1 mg/kg.All siRNAs were synthesized by Alnylam and were characterized byelectrospray mass spectrometry and anion exchange HPLC. The sequencesfor the sense and antisense strands of FVII, ApoB, and Control siRNAshave been reported (Akinc, A. et al., Nat. Biotechnol 26:561-569(2008)). The sequences for the sense and antisense strands of the TTRsiRNA were as follows:

(SEQ ID NO: 37)  siTTR sense: 5′-GuAAccAAGAGuAuuccAuTT-3′; and(SEQ ID NO: 38) siTTR antisense: 5′-AUGGAAuACUCUUGGUuACTT-3′, with 2′-O-Me modified nucleotides shown in lower case. siRNAs weregenerated by annealing equimolar amounts of complementary sense andantisense strands.

Tissues were harvested at 48 hours post-administration, and liver mRNAlevels of TTR were determined. A clear dose response was obtained withan apparent ED₅₀ of ˜0.3 mg/kg (FIG. 8B). A toxicological analysisindicated that the treatment was well tolerated at the dose levelstested, with no treatment-related changes in animal appearance orbehavior. No dose-dependent, clinically significant alterations in keyclinical chemistry or hematological parameters were observed (Table 13).

TABLE 13 Clinical chemistry, and hematology parameters in NHPs siRNATotal dose ALT AST Bilirubin BUN RBC Hemoglobin WBC PLT Treatment(mg/kg) (U/L) (U/L) (mg/dL) (mg/dL) (×10⁶/μL) (g/dL) (×10³/μL) (×10³/μL)PBS — 54 ± 25 51 ± 27 0.3 ± 0.1 27 ± 4 4.6 ± 0.5 13.8 ± 0.6 17.6 ± 3.0515 ± 70 siApoB 1 42 ± 11 49 ± 12 0.3 ± 0.1 23 ± 3 6.0 ± 0.2 14.2 ± 0.913.6 ± 3.7 508 ± 49 siTTR 0.03 57 ± 11 47 ± 12 0.1 ± 0   15 ± 4 4.8 ±0.4 11.5 ± 0.9 10.9 ± 2.2  495 ± 105 siTTR 0.1 50 ± 22 63 ± 47 0.13 ±0.1  20 ± 3 5.0 ± 0.0 11.1 ± 0.4 12.9 ± 3.3 528 ± 22 siTTR 0.3 67 ± 3866 ± 18 0.1 ± 0   21 ± 6 5.1 ± 0.2 11.0 ± 0.5 11.1 ± 5.5 529 ± 72 siTTR1 47 ± 5  43 ± 7  0.13 ± 0.1  19 ± 1 4.9 ± 0.1 10.9 ± 0.4 10.7 ± 1.6 477± 34

In summary, a rational design approach was employed for the discovery ofnovel lipids for use in next-generation LNP systems to deliver RNAitherapeutics. Using this approach, important structure-activityconsiderations for ionizable cationic lipids were described, andmultiple lipids based on the DLinDMA structure were designed andcharacterized. A SNALP formulation of the best performing lipid(DLin-K-C2-DMA) was well-tolerated in both rodent and non-human primatesand exhibited in vivo activity at siRNA doses as low as 0.01 mg/kg inrodents, as well as silencing of a therapeutically significant gene(TTR) in non-human primates. Notably, the TTR silencing achieved in thiswork (ED₅₀˜0.3 mg/kg), represents a significant improvement in activityrelative to previous reports of LNP-siRNA mediated silencing innon-human primates. The efficacy observed in this study, to ourknowledge, represents the highest level of potency observed for an RNAitherapeutic in non-human primates to date, and highlights theconsiderable progress that has been made in both RNAi and deliverytechnologies.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet, areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

The invention claimed is:
 1. An amino lipid having the structure: